Method for producing 4-hydroxy-L-isoleucine

ABSTRACT

A highly active L-isoleucine dioxygenase from  Bacillus thuringiensis  is provided. A method for manufacturing (2S,3R,4S)-4-hydroxy-L-isoleucine or a salt thereof by reacting L-isoleucine in an aqueous solvent in the presence of L-isoleucine dioxygenase and isolating (2S,3R,4S)-4-hydroxy-L-isoleucine is also provided.

This application is a divisional under 35 U.S.C. §120 to U.S. patentapplication Ser. No. 12/412,823, filed Mar. 27, 2009now U.S. Pat. No.8,114,651, which was a continuation under 35 U.S.C. §120 to PCT PatentApplication No. PCT/JP2007/069520, filed on Sep. 28, 2007, which claimedpriority under 35 U.S.C. §119 to Japanese Patent Application No.2006-265452, filed Sep. 28, 2006, U.S. Provisional Patent ApplicationNo. 60/829,577, filed on Oct. 16, 2006, Japanese Patent Application No.2006-345461, filed Dec. 22, 2006, and Russian Patent Application No.2007104645, filed on Feb. 7, 2007, all of which are incorporated byreference. The Sequence Listing filed electronically herewith is alsohereby incorporated by reference in its entirety (File Name:2012-01-05T_US-312D_Seq_List; File Size: 54 KB; Date Created: Jan. 5,2012)

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to the microbiological industry, andspecifically to a novel dioxygenase and methods for manufacturing4-hydroxy-L-isoleucine or a salt thereof.

2. Background Art

4-hydroxy-L-isoleucine is an amino acid which can be extracted andpurified from fenugreek seeds (Trigonella foenum-graecum L.leguminosae). 4-Hydroxy-L-isoleucine has an insulinotropic activity, andis of great interest because its stimulating effect is clearly dependenton plasma glucose concentrations, as demonstrated both in isolatedperfused rat pancreas and human pancreatic islets (Sauvaire, Y. et al,Diabetes, 47: 206-210, (1998)). The only class of insulinotropic drugscurrently used to treat type II diabetes, or non-insulin-dependentdiabetes (NIDD) mellitus (NIDDM), are sulfonylureas, and these drugs donot demonstrate this glucose dependency (Drucker, D. J., Diabetes 47:159-169, (1998). As a result, hypoglycemia remains a common undesirableside effect of sulfonylurea treatment (Jackson, J., and Bessler, R.Drugs, 22: 211-245; 295-320, (1981); Jennings, A. et al. Diabetes Care,12: 203-208, (1989)). Methods for improving glucose tolerance are alsoknown (Am. J. Physiol. Endocrinol., Vol. 287, E463-E471, 2004).Enhancing glucometabolism activity, and the potential application ofthis activity in pharmaceuticals and health foods, has been reported(Japanese Patent Application Laid-Open No. Hei 6-157302,US2007-000463A1).

4-hydroxy-L-isoleucine, which is only found in plants, might beconsidered for the treatment of type II diabetes due to its particularinsulinotropic action, since this is a disease characterized bydefective insulin secretion associated with various degrees of insulinresistance (Broca, C. et al, Am. J. Physiol. 277 (Endocrinol. Metab.40): E617-E623, (1999)).

Methods of oxidizing iron, ascorbic acid, 2-oxyglutaric acid, andoxygen-dependent isoleucine by utilizing dioxygenase activity infenugreek extract has been reported for manufacturing4-hydroxy-L-isoleucine (Phytochemistry, Vol. 44, No. 4, pp. 563-566,1997). However, this method is unsatisfactory for manufacturing4-hydroxy-L-isoleucine because the activity of the enzyme is inhibitedby isoleucine concentrations of 20 mM and above, the enzyme has not beenidentified, the enzyme is derived from plant extracts and it isdifficult to obtain large quantities, and the enzyme is unstable.

An efficient eight-step synthesis of optically pure(2S,3R,4S)-4-hydroxyisoleucine with a 39% overall yield has beendisclosed. The key steps of this synthesis involve the biotransformationof ethyl 2-methylacetoacetate to ethyl(2S,3S)-2-methyl-3-hydroxy-butanoate with Geotrichum candidum and anasymmetric Strecker synthesis (Wang, Q. et al, Eur. J. Org. Chem.,834-839 (2002)).

A short six-step chemoenzymatic synthesis of(2S,3R,4S)-4-hydroxyisoleucine while controlling the stereochemistry,the last step being the enzymatic resolution by hydrolysis of aN-phenylacetyl lactone derivative using commercially availablepenicillin acylase G immobilized on Eupergit C(E-PAC), has also beendisclosed (Rolland-Fulcrand, V. et al, J. Org. Chem., 873-877 (2004)).

But currently, the cloning of any L-isoleucine dioxygenase has not beenreported, nor of its use for producing (2S,3R,4S)-4-hydroxy-L-isoleucineby direct enzymatic hydroxylation of L-isoleucine.

As for production of isoleucine analogues by microorganisms, productionof 2-amino-3-keto-4-methylpentanoic acid (AMKP) by Bacillus bacteria hasbeen reported (Bioorganic Chemistry, Vol. 6, pp. 263-271 (1977)).However, there are no reports about isoleucine hydroxylases derived frommicroorganisms.

SUMMARY OF THE INVENTION

An aspect of the present invention is to provide a method for producing4-hydroxyisoleucine (which includes both the free form and salt forms f,and may referred to as “4HIL”) by using an enzyme derived from amicroorganism, which can be prepared in large amounts.

Aspects of the present invention include providing a microorganismhaving an enzymatic activity for producing 4-hydroxyisoleucine fromisoleucine, and a method for producing 4-hydroxyisoleucine fromisoleucine via a hydroxylation reaction using an enzyme derived from amicroorganism. The above aspects were achieved by isolating amicroorganism having an enzymatic activity for producing4-hydroxyisoleucine from isoleucine.

An aspect of present invention is to enhance production of(2S,3R,4S)-4-hydroxy-L-isoleucine (which includes both the free form andsalt forms, and may be referred to as “(2S,3R,4S)-4HIL”), and to providea method for manufacturing (2S,3R,4S)-4-hydroxy-L-isoleucine or a saltthereof by direct enzymatic hydroxylation of L-isoleucine usingL-isoleucine dioxygenase or a bacterium having the L-isoleucinedioxygenase activity. As a result of extensive research conducted inconsideration of the aforementioned problems, the inventors of thepresent invention isolated from nature a bacterium having a high levelof L-isoleucine dioxygenase activity, cloned the gene encodingL-isoleucine dioxygenase, and found that the L-isoleucine dioxygenasemay be used in the synthesis of the (2S,3R,4S)-4-hydroxy-L-isoleucine.

Namely, it is an aspect of the present invention to provide L-isoleucinedioxygenase and the DNA encoding L-isoleucine dioxygenase, and a methodfor producing (2S,3R,4S)-4-hydroxy-L-isoleucine using the L-isoleucinedioxygenase. The above aspects were achieved by finding the novelL-isoleucine dioxygenase.

It is an aspect of the present invention to provide a method forproducing 4-hydroxyisoleucine or a salt thereof, comprising A)subjecting isoleucine or a salt thereof to a hydroxylation reaction inthe presence of a hydroxylase derived from a microorganism to produce areaction product, and B) isolating 4-hydroxyisoleucine or a salt thereoffrom the reaction product.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the microorganism belongs to the genusBacillus.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the microorganism is selected from the groupconsisting of Bacillus thuringiensis, Bacillus licheniformis, Bacillussphaericus, Bacillus cereus, and Bacillus weihenstephanensis.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said hydroxylase is present in a cell lysateprepared from microbial cells which were in a logarithmic growth phase.

It is a further aspect of the present invention to provide the method asdescribed above, wherein L-isoleucine is subjected to hydroxylation.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the hydroxylase is a dioxygenase.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the hydroxylase has the following properties:

(a) requires oxygen, Fe²⁺, ascorbic acid, and 2-oxoglutaric acid ascofactors,

(b) has an optimum reaction pH of 5 to 8,

(c) has an optimum reaction temperature of 45° C. or lower,

(d) is inactivated at 50° C. or higher, and

(e) is inhibited by EDTA, Cu²⁺, and Zn²⁺.

It is a further aspect of the present invention is to provide adioxygenase which has the following properties and is able to beisolated from a Bacillus bacterium:

(a) requires oxygen, Fe²⁺, ascorbic acid, and 2-oxoglutaric acid ascofactors,

(b) has an optimum reaction pH of 5 to 8,

(c) has an optimum reaction temperature of 45° C. or lower,

(d) is inactivated at 50° C. or higher,

(e) is inhibited by EDTA, Cu²⁺ and Zn²⁺.

(f) comprises subunits having a molecular weight of 29,000±2,000 asmeasured by sodium dodecylsulfate-polyacrylamide gel electrophoresis,and

(g) comprises the amino acid sequence of SEQ ID NO: 5 at the N-terminus.

It is a further aspect of the present invention is to provide thedioxygenase as described above, wherein the Bacillus bacterium isBacillus thuringiensis.

It is a further object of the present invention to provide a DNAselected from the group consisting of:

A) a DNA comprising the nucleotide sequence of SEQ ID No:1;

B) a DNA that hybridizes under stringent conditions with a DNAcomprising a nucleotide sequence which is complementary to thenucleotide sequence of SEQ ID No:1, and encodes a protein havingL-isoleucine dioxygenase activity;

C) a DNA that encodes a protein comprising the amino acid sequence ofSEQ ID No:2;

D) a DNA that encodes a protein having the amino acid sequence of SEQ IDNO: 2, but which includes one or several substitutions, deletions,insertions, additions, or inversions of one or several amino acidresidues, and has L-isoleucine dioxygenase activity; and

E) a DNA that encodes a protein comprising an amino acid sequence thatis at least 98% homologous to the amino acid sequence of SEQ ID No:2,and has L-isoleucine dioxygenase activity.

It is a further aspect of the present invention to provide a recombinantDNA obtained by ligating the DNA containing the same with a vector DNA.

It is a further aspect of the present invention to provide a celltransformed with the recombinant DNA containing the same.

It is a further aspect of the present invention to provide a process forproducing a protein having L-isoleucine dioxygenase activity, theprocess comprising: A) cultivating the cell containing the samerecombinant DNA in a medium, and collecting the protein withL-isoleucine dioxygenase activity from the medium, cells or both.

It is a further aspect of the present invention to provide a proteinselected from the group consisting of:

(f) a protein comprising the amino acid sequence of SEQ ID No: 2;

(g) a protein having the amino acid sequence of SEQ ID NO: 2, but whichincludes one or several substitutions, deletions, insertions, additions,or inversions of one or several amino acids and has L-isoleucinedioxygenase activity; and

(h) a protein that is at least 98% homologous to the amino acid sequenceof SEQ ID No: 2 and has L-isoleucine dioxygenase activity.

It is a further object of the present invention to provide a proteincomprising:

(A) the ability to catalyze the production of(2S,3R,4S)-4-hydroxy-L-isoleucine by hydroxylation of L-isoleucine;

(B) an activity which is dependent on Fe²⁺; and

(C) a molecular weight per subunit as measured by SDS-PAGE of about29±2.0 kDa.

It is a further aspect of the present invention to provide a method formanufacturing (2S,3R,4S)-4-hydroxy-L-isoleucine or a salt thereof, themethod comprising the steps of:

(A) placing L-isoleucine in an aqueous solvent in the presence of anL-isoleucine dioxygenase selected from the group consisting of:

(a) a protein comprising an amino acid sequence selected from the groupconsisting of SEQ ID No: 2, 8, 13, 17, and 21,

(b) a protein comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 8, 13, 17, and 21, but which includes one orseveral substitutions, deletions, insertions, additions, or inversionsof one or several amino acids and has L-isoleucine dioxygenase activity,

(c) a protein that is at least 70% homologous to an amino acid sequenceselected from the group consisting of SEQ ID No: 2, 8, 13, 17, and 21,and has L-isoleucine dioxygenase activity, and

(d) combinations thereof;

(B) isolating (2S,3R,4S)-4-hydroxy-L-isoleucine from the aqueoussolvent.

It is a further aspect of the present invention to provide a method formanufacturing (2S,3R,4S)-4-hydroxy-L-isoleucine or a salt thereof,comprising the steps of:

(A) placing L-isoleucine in an aqueous solvent comprising a bacterialproduct comprising an L-isoleucine dioxygenase selected from the groupconsisting of:

(a) a protein comprising an amino acid sequence selected from the groupconsisting of SEQ ID No: 2, 8, 13, 17, and 21,

(b) a protein comprising an amino acid sequence selected from the groupconsisting of SEQ ID No: 2, 8, 13, 17, and 21, but which includes one orseveral substitutions, deletions, insertions, additions, or inversionsof one or several amino acids, and has L-isoleucine dioxygenaseactivity, and

(c) a protein that is at least 70% homologous to an amino acid sequenceselected from the group consisting of SEQ ID No: 2, 8, 13, 17, and 21and has L-isoleucine dioxygenase activity, and

(d) combinations thereof; and

(B) isolating (2S,3R,4S)-4-hydroxy-L-isoleucine from the aqueoussolvent.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said activity of the L-isoleucine dioxygenaseis enhanced by modifying the bacterium.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the activity of the L-isoleucine dioxygenase isenhanced by increasing the expression of the L-isoleucine dioxygenase.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the expression of the L-isoleucine dioxygenaseis increased by a method selected from the group consisting of:

A) modifying an expression control sequence of the gene encoding theL-isoleucine dioxygenase,

B) increasing the copy number of the gene encoding the L-isoleucinedioxygenase, and

C) combinations thereof.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium belongs to a genus selected fromthe group consisting of Escherichia, Pseudomonas, Corynebacterium,Arthrobacter, Aspergillus and Bacillus.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium is selected from the groupconsisting of Escherichia coli, Arthrobacter simplex, Corynebacteriumglutamicum, Arthrobactor globiformis, Arthrobactor sulfureus,Arthrobactor viscosus and Bacillus subtilis.

It is a further object of the present invention to provide the method asdescribed above, wherein the bacterial product is selected from thegroup consisting of a bacterial culture, cells, treated cells and celllysate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which shows the accumulation over time of4-hydroxyisoleucine and AMKP which are produced in the culture of the2-e-2 strain.

FIG. 2 is a graph which shows the change in turbidity over time of theculture of the 2-e-2 strain.

FIG. 3 is a graph which shows the accumulation over time of4-hydroxyisoleucine and AMKP using resting cells of the 2-e-2 strain.

FIG. 4 is a graph which shows the 4-hydroxyisoleucine production invarious samples.

FIG. 5 is a graph which shows the relative pH dependency of the4-hydroxyisoleucine production in the 2-e-2 cell lysate.

FIG. 6 is a graph which shows the relative temperature dependency of the4-hydroxyisoleucine production in the 2-e-2 cell lysate.

FIG. 7 is a graph which shows the relative temperature stability of the4-hydroxyisoleucine production in the 2-e-2 cell lysate.

FIG. 8 is a graph showing the pH dependency of the 4-hydroxyisoleucineproduction using a purified enzyme.

FIG. 9 is a graph showing the temperature dependency of the4-hydroxyisoleucine production using a purified enzyme.

FIG. 10 is a graph which shows the temperature stability of the4-hydroxyisoleucine production using a purified enzyme.

FIG. 11 is a flowchart of the process for producing IDO.

FIG. 12 is a photograph of the SDS-PAGE gel. The lanes represent theprotein preparation at the various stages of IDO purification fromBacillus. Lanes: 1—molecular weight standards; 2—crude cell lysate;3—ammonium sulphate precipitation; 4—SEC; 5—AEC.

FIG. 13 shows the MS-identification of IDO from Bacillus thuringiensis2-e-2. Peptide sequences in the far right column of the box are listedin, in order, SEQ ID NOs:22-23.

FIG. 14 shows the putative translation start of the Bacillusthuringiensis (serovar israelensis, ATCC 35646) RBTH_(—)06809 ORF. Thenucleotide sequence is shown in SEQ ID NO:34. The amino acid sequence isshown in SEQ ID NO:35.

FIGS. 15A, B and C show the artificial expression modules of therecombinant pMW119-IDO(Lys, 32/23) plasmids. In FIG. 15A, the nucleotidesequence is shown in SEQ ID NO:3 and amino acid sequences are shown inSEQ ID NOs:36-37. In FIG. 15B, the nucleotide sequence is shown in SEQID NO:38 and amino acid sequences are shown in SEQ ID NOs:39-40. In FIG.15C, the nucleotide sequence is shown in SEQ ID NO:41 and the amino acidsequences are shown in SEQ ID NOs:42 and 40.

FIG. 16 is a physical map of the pMW119-IDO(Lys, 23) plasmid and the DNAsequence of the cloned BamHI-SacI fragment containing the IDO structuralgene. The spontaneous point mutation in the regulatory region is markedby shading. The nucleotide sequence is shown in SEQ ID NO:43. The aminoacid sequence is shown in SEQ ID NO:44.

FIG. 17 is a physical map of the pMW119-IDO(Lys, 32) plasmid, and theDNA sequence of the cloned BamHI-SacI fragment containing the IDOstructural gene. The nucleotide sequence is shown in SEQ ID NO:45. Theamino acid sequence is shown in SEQ ID NO:46.

FIG. 18 shows the DNA alignment of the structural genes corresponding toIDO(Lys, 23), IDO(Lys, 32) and RBTH_(—)6809 (5′-end truncated). Variablepositions are marked by shading. The nucleotide sequence of IDO (Lys,RBTH) is shown in SEQ ID NO:47. The nucleotide sequence of IDS (Lys, 23)is shown in SEQ ID NO:49. The nucleotide sequence of IDO (Lys, 32) isshown in SEQ ID NO:7.

FIG. 19 shows the protein alignment of the IDO(Lys, 23), IDO(Lys, 32),and RBTH_(—)06890 ORF. Variable positions are marked by shading. Theamino acid sequence of IDO (Lys, RBTH) is shown in SEQ ID NO:48. Theamino acid sequence of IDO (Lys, 23) is shown in SEQ ID NO:2. The aminoacid sequence of IDO (Lys, 32) is shown in SEQ ID NO:8.

FIG. 20 shows the protein alignment of IDO from Bacillus thuringienesis,BC1061 from Bacillus cereus, and the conserved hypothetical protein fromBacillus weihenstephanensis. The amino acid sequence of Bacillus cereusis shown in SEQ ID NO:50. The amino acid sequence of Bacillusthuringiensis is shown in SEQ ID NO:48. The amino acid sequence ofBacillus weihenstephanensis is shown in SEQ ID NO:51. The amino acidsequence of Ile hydroxylase is shown in SEQ ID NO:6.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The name “4-hydroxyisoleucine” may indicate one diasteromer, or amixture of two or more diastereomers, such as(2S,3S,4S)-4-hydroxyisoleucine, (2S,3R,4R)-4-hydroxyisoleucine,(2S,3S,4R)-4-hydroxyisoleucine, and (2S,3R,4S)-4-hydroxyisoleucine.4-Hydroxyisoleucine is preferably (2S,3R,4S)- or(2R,3R,4S)-4-hydroxyisoleucine or a mixture thereof, and more preferably(2S,3R,4S)-4-hydroxyisoleucine.

In particular, the term “(2S,3R,4S)-4-hydroxy-L-isoleucine” or“(2S,3R,4S)-4HIL” may refer to single chemical compound or a mixturecontaining at least (2S,3R,4S)-4-hydroxyisoleucine.

The term “bacterium” or “microorganism” includes enzyme-producingbacteria or microorganisms, or a mutant or genetic recombinant of suchbacteria or microorganisms in which the targeted enzymatic activity ispresent or has been enhanced, and the like.

<I> Enzymatic Activity which can Catalyze Production of 4HIL fromIsoleucine

A hydroxylase is used as the catalyst in the described methods forproducing 4-hydroxyisoleucine from isoleucine, and this hydroxylase maybe present in a microbial culture, the bacterial cells, or a celllysate. The hydroxylase may also be in purified form so long as theenzymatic activity of converting isoleucine to 4-hydroxyisoleucine ismaintained. Disrupted cells containing crude enzyme, or compositionscontaining somewhat purified enzyme may also be used.

Examples of the hydroxylase include oxygenases, dioxygenases, and soforth, and dioxygenases are preferred. A hydroxylase that can beisolated from the 2-e-2 bacterial strain and has the followingproperties is more preferred:

(a) requires oxygen, Fe²⁺, ascorbic acid, and 2-oxoglutaric acid ascofactors,

(b) has an optimum reaction pH of 5 to 8,

(c) has an optimum reaction temperature of 45° C. or lower,

(d) is inactivated at 50° C. or higher, and

(e) is inhibited by EDTA, Cu²⁺ and Zn²⁺.

The hydroxylase from the 2-e-2 strain has the following additionalproperties:

(f) is made up of subunits having a molecular weight of 29,000±2,000 asmeasured by sodium dodecylsulfate polyacrylamide gel electrophoresis,

(g) has the amino acid sequence of SEQ ID NO: 5 at the N-terminus.

When the hydroxylase requires a cofactor, it is preferable to add orsupply the cofactor to the system. Examples of the cofactor fordioxygenase include, for example, Fe²⁺, ascorbic acid, and2-ketoglutaric acid. These cofactors may be added or supplied to thesystem as a salt when possible.

Any microorganism can be used so long as the enzymatic activity whichcatalyzes the conversion of isoleucine to 4-hydroxyisoleucine under theconditions necessary for hydroxylation is present.

Examples of the microorganism may include microorganisms belonging tothe genus Bacillus or Pseudomonas, mutants or derivatives thereof, andso forth. Furthermore, the microorganism may be one in which thehydroxylase is introduced and expressed by genetic recombination, andwhich is able to produce 4-hydroxyisoleucine.

Specific examples include Bacillus thuringiensis (strains 2-e-2, AKU238, NBRC 3958, ATCC 35646, etc.), Bacillus licheniformis (strains AKU223, IAM 11054, etc.), Bacillus sphaericus (strains AKU 227, NBRC 3526,etc.), Bacillus cereus strain ATCC 14579, and Bacillusweihenstephanensis strain KBAB4. The 2-e-2 strain (AJ110584) wasdeposited at the independent administrative agency, National Instituteof Advanced Industrial Science and Technology, International PatentOrganism Depositary (Tsukuba Central 6, 1-1, Higashi 1-Chome,Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) under the provisions of theBudapest Treaty on Sep. 27, 2006, and given an accession number of FERMBP-10688.

Strains with a name beginning with AKU can be obtained from theLaboratory of Fermentation Physiology and Applied Microbiology, Divisionof Applied Life Sciences, Graduate School of Agriculture, KyotoUniversity. Strains with a name beginning with IAM are maintained at theIAM culture collection, Laboratory of Bioresources, Institute ofMolecular and Cellular Biosciences, the University of Tokyo, and can beobtained using their registration numbers. The registration numberscorresponding to the strains are listed in the IAM catalog (IAMCatalogue of Strains Third Edition, 2004). Strains with a name beginningwith NBRC can be obtained from the independent administrative agency,National Institute of Technology and Evaluation (2-5-8, Kazusa Kamatari,Kisarazu-shi, Chiba, 292-0818). Strains with a name beginning with ATCCcan be obtained from the American Type Culture Collection (ATCC) (Postaladdress: ATCC, P.O. Box 1549, Manassas, Va. 20108, 1, United States ofAmerica). Bacillus weihenstephanensis strain KBAB4 can be obtained fromthe Institut National de la Recherche Agronomique (Postal address:Genetique Microbienne, INRA, Domaine de Vilvert, 79352 Jouy en Josascedex, France).

The Bacillus thuringiensis 2-e-2 strain has been newly isolated from thesoil in Kyoto, and the scientific properties of this strain are shownbelow.

Taxonomic properties of Bacillus thuringiensis 2-e-2 strain:

1. Phenotype

Cell morphology: Bacillus (size: 1.0 to 1.2×2.0 to 3.0 μm)

Gram staining: +

Endospore: +

Attitude to oxygen: Aerobic

Favorable growth at 20 to 35° C.

Optimum pH: pH 7.0 to 7.5

2. Molecular phylogenetic analysis based on the nucleotide sequence ofthe 16S rDNA

To determine the 16S rDNA nucleotide sequence of the 2-e-2 strain (SEQID NO: 9), searches were conducted on the bacterial strain database(NCIMB Japan, Shizuoka) and several international nucleotide sequencedatabases (GenBank/DDBREMBL), and BLAST, and the 30 most homologousstrains were determined. Then, a molecular phylogenic tree was createdusing the 16S rDNA nucleotide sequences for the 30 homologous strainsretrieved from the bacteria strain database according to theneighbor-joining method. For the homology search and creating asimplified molecular phylogenetic tree, DNAsisPro was used (HitachiSoftware Engineering Co., Ltd., Tokyo).

As a result of the homology search of the bacterial strain database andBLAST, a partial nucleotide sequence of the 16S rDNA of the 2-e-2 strainwas a 100% match with that of the 16S rDNA of the Bacillus thuringiensisATCC 10792 strain. As a result of the homology search ofGenBank/DDBREMBL, the 16S rDNA of the 2-e-2 strain showed a highhomology with that of Bacillus thuringiensis. Furthermore, the 2-e-2strain is found on substantially the same phylogenetic branch as that ofthe 16S rDNA of Bacillus thuringiensis, which demonstrates that they arevery closely related.

3. Results of Classification and Identification

The 2-e-2 strain appeared to be Bacillus bacteria based on themorphological observations, and the analysis of the partial 16S rDNAsequence also showed that the 2-e-2 strain belongs to Bacillusthuringiensis. Since no microorganisms have been reported that have AMKPproduction activity at the same level as the 2-e-2 strain, this strainwas identified as a novel strain.

Known and typical culture methods can be used for culturing themicroorganisms. Either a natural or synthetic medium may be used for theculture so long as the medium contains a carbon source, nitrogen source,inorganic salts, and so forth that can be assimilated by themicroorganism, and the microorganism can be efficiently cultured in themedium.

The carbon source may be one that can be assimilated by themicroorganism, and includes saccharides such as glucose, fructose,sucrose, maltose, starch, starch hydrolysate, and molasses, organicacids such as acetic acid, lactic acid, and gluconic acid, and alcoholssuch as ethanol and propanol. As the nitrogen source, ammonia, ammoniumsalts of various inorganic acids, and organic acids such as ammoniumsulfate, ammonium chloride, ammonium acetate, and ammonium phosphate,other nitrogen-containing compounds, peptone, meat extract, yeastextract, corn steep liquor, casein hydrolysate, soybean meal, soybeanmeal hydrolysate, various fermenting cells, and digestion productsthereof, and so forth can be used so long as the microorganism canassimilate the chosen source.

As the inorganic salts, potassium phosphate, ammonium sulfate, ammoniumchloride, sodium chloride, magnesium sulfate, ferrous sulfate, manganesesulfate, and so forth can be used, so long as the chosen microorganismcan utilize the chosen salt. In addition, salts of calcium, zinc, boron,copper, cobalt, molybdenum, and so forth may be added as trace elements.Furthermore, vitamins such as thiamin and biotin, amino acids such asglutamic acid and aspartic acid, nucleic acid-related substances such asadenine and guanine, and so forth may be added as required.

The culture is performed under aerobic conditions such as by shaking ordeep aeration stirring. The culture temperature is preferably 10 to 37°C., and the culture time is 5 to 40 hours. The pH of the culture ismaintained at 5.0 to 9.0 during the culture. The pH is adjusted by usinginorganic or organic acids, alkaline solutions, urea, calcium carbonate,ammonia, or the like.

The crude enzyme may be present in disrupted cells, i.e., a microbialcell lysate. The lysate may contain extracellular hydroxylase, andexamples of the lysate include bacterial cells treated with surfactant,organic solvent, or enzyme(s), bacterial cells subjected toultrasonication, mechanical disruption, or a solvent treatment, afraction of cellular proteins, a solidified product of processed cells,and so forth. The cell lysate is preferably prepared from cells in thelogarithmic growth phase.

To prepare a cell lysate, cultured bacterial cells can be washed with anisotonic solution such as physiological saline, and then disrupted byany means, for example, by compression disruption using a French press,glass beads, ultrasonic disruptor, Manton Gaulin homogenizer, mortar, acombination thereof, or the like. For more efficient cell disruption,cell membrane surfaces may be physically or chemically treated byfreezing, treated with enzymes, or the like. During the cell disruption,cells are always maintained at a low temperature, and when the celllysis temperature is raised due to the disruption process, thetemperature may be immediately lowered.

Examples of aqueous medium which may be used for the cell lysateinclude, but are not limited to, water and buffers such as those ofborate, acetate, carbonate, Tris, phosphate, citrate, and Good buffer.Furthermore, glycerol, DTT or the like may be added as an enzymestabilizer, and EDTA, EGTA, PMSF, pepstatin, E-64 or the like may beadded as a protease inhibitor. A combination of inhibitors, an inhibitorcocktail, or the like may also be added.

When using a cell lysate in the production of 4-hydroxyisoleucine, thecomposition of the substrate solution may be 100 mM HEPES buffer (pH7.0) containing 5 mM isoleucine, 5 mM 2-ketoglutaric acid, 5 mM ascorbicacid, and 5 mM Fe²⁺ in a volume of 100 μl, and the reaction is performedat 30° C. for 60 minutes. Other than the HEPES buffer, other bufferssuch as MES buffer and GTA wide range buffer may also be used. After therequired enzyme inactivation, the centrifugation supernatant fractionsolution is filtered, and production of 4-hydroxyisoleucine is measuredby high performance liquid chromatography or TLC.

4-Hydroxyisoleucine may be quantified by any method so long as thechosen analysis system can separate 4-hydroxyisoleucine from othercomponents, and examples include TLC and high performance liquidchromatography. High performance liquid chromatography is preferred forquantitative analysis because of its high sensitivity and superiorseparating ability. Examples include the Waters AccQ-Tag™ method, whichis an amino acid analysis method, and so forth. By a modified WatersAccQ-Tag™ method (see the examples described later), diastereomers of4-hydroxyisoleucine can be separated, and naturally occurring4-hydroxyisoleucine and 2-amino-3-methyl-4-ketopentanoic acid, which isa keto compound formed by oxidation of the hydroxyl group of4-hydroxyisoleucine, can be separated.

4-Hydroxyisoleucine can be isolated by using any common amino acidpurification method. For example, by using a combination of an ionexchange resin, a membrane, crystallization, and so forth,4-hydroxyisoleucine can be isolated from the supernatant after solidsare removed by centrifugation.

The pH for the isoleucine hydroxylation reaction is preferably 5 to 8.Factors which are essential for the enzymatic reaction are preferablypresent in the reaction mixture. When Fe²⁺ is essential, a reactionmixture composition should be used which is unlikely to cause chelationwith the Fe²⁺, such as HEPES buffer, MES buffer, and GTA wide rangebuffer. However, the reaction mixture composition is not limited so longas the action of Fe²⁺ is maintained.

The temperature for the hydroxylation reaction of isoleucine is usually15 to 30° C., preferably 45° C. or lower. The reaction time is usually 5minutes to 200 hours, although it varies depending on the amount of theenzyme.

The L-isoleucine form is preferably used in the hydroxylation reaction.

Examples of the solvent in which the reaction can be performed includeaqueous solvents, for example, water, buffers such as carbonate,acetate, borate, citrate and Tris, organic solvents, for example,alcohols such as methanol and ethanol, esters such as ethyl acetate,ketones such as acetone, amides such as acetamide, aqueous solventscontaining these organic solvents, and so forth. Furthermore, a factorfor activating the hydroxylation reaction may be added as required.

The protein concentration of the cell lysate is 0.1 to 50 mg/ml,preferably 0.5 to 20 mg/ml (in terms of cell weight (wet weight)).4-Hydroxy-L-isoleucine can be produced by adding the cell lysate,substrate, and cofactors to an aqueous medium at suitable concentrationsand allowing the reaction to proceed at a temperature of 45° C. orlower, preferably 30° C., and pH 5 to 12, preferably pH 5 to 7.5, for 5minutes to 120 hours.

<II> L-Isoleucine Dioxygenase and DNA Encoding the L-IsoleucineDioxygenase, and their Uses

The following is a detailed explanation of [I] L-isoleucine dioxygenase,and [II] a process for producing (2S,3R,4S)-4-hydroxy-L-isoleucine usingL-isoleucine dioxygenase with reference to the accompanying drawings.

[I] L-Isoleucine Dioxygenase

Within the Bacillus genus, bacterial strains were found which containedL-isoleucine dioxygenase, and which were able to form (2S,3R,4S)-4HIL.L-Isoleucine dioxygenase is also referred to as “IDO”.

As described above, the unique microbe Bacillus thuringiensis strain2-e-2 was found by screening environmental microorganisms. This strainis able to catalyze the reaction in which (2S,3R,4S)-4HIL is directlyformed from L-isoleucine. The term “L-isoleucine” refers to both thefree form and the salt form. The novel L-isoleucine dioxygenase waspurified and isolated from the cultivated microbial cells, and ishereinafter abbreviated as “IDO(Lys,23)”.

Furthermore, the N-terminal amino acid sequence of IDO(Lys,23) wasdetermined by purifying the dioxygenase from Bacillus thuringiensis2-e-2 strain. Bacillus thuringiensis 2-e-2 was named Bacillusthuringiensis AJ110584 and deposited at the International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566,Japan) on Sep. 27, 2006 and given an accession number of FERM BP-10688under the provisions of Budapest Treaty.

Furthermore, DNA molecules of about 30 base pairs deduced from the aminoacid sequences of the IDO(Lys,23) were synthesized. Then, the entirelength of the DNA that encodes IDO(Lys,23) was isolated usingchromosomal DNA from Bacillus thuringiensis strain 2-e-2. TheL-isoleucine dioxygenase from Bacillus thuringiensis (serovarisraelensis) strain (ATCC 35646), “IDO(Lys,32)”, was used as thecontrol. These DNA molecules were used to construct recombinant plasmidsand transform E. coli cells. Subsequent analysis of the recombinant E.coli IDO activity revealed a much higher (5 times) activity ofIDO(Lys,23) in comparison with IDO(Lys,32). It appears that thisdifference in activity is due to the unique mutations present in theIDO(Lys,23) gene from Bacillus thuringiensis strain 2-e-2.

The DNA encoding IDO(Lys,23) is shown in SEQ ID No: 1. Furthermore, theamino acid sequence of IDO(Lys,23) encoded by the nucleotide sequence ofSEQ ID NO: 1 is shown in SEQ ID No: 2. IDO(Lys,23) of SEQ ID NO: 2possesses the L-isoleucine dioxygenase activity, and catalyzes thereaction in which (2S,3R,4S)-4HIL shown in the following formula (I) isdirectly synthesized from one molecule of L-isoleucine.

Next, a detailed explanation is provided of (1) a DNA encodingL-isoleucine dioxygenase, (2) properties of L-isoleucine dioxygenase,and (3) a process for producing L-isoleucine dioxygenase.

(1) A DNA Encoding L-isoleucine Dioxygenase

The IDO(Lys,23) gene having the nucleotide sequence of SEQ ID No: 1 wasisolated from the chromosomal DNA of Bacillus thuringiensis 2-e-2 asdescribed in the Examples section. The nucleotide sequence of SEQ ID No:1 from Bacillus thuringiensis 2-e-2 encoding IDO(Lys,23) demonstratesthe high level of homology both at the nucleotide (FIG. 19) and aminoacid levels (FIG. 18) with the non-annotated part of the genomicnucleotide sequence from Bacillus thuringiensis (serovar israelensis)(ATCC 35646), one copy of which was submitted 17-JAN-2007 to theNational Center for Biotechnology Information, NIH, Bethesda, Md. 20894,USA (Accession No: AAJM00000000.1, GI:74494335), and the other wasobtained as a result of sequencing the genomic DNA encoding IDO(Lys,32)of the same Bacillus thuringiensis (serovar israelensis) strain receivedfrom Russian National Collection of Industrial Microorganisms (VKPM)stored under accession number VKPM B-197. The nucleotide sequence fromBacillus thuringiensis (serovar israelensis) strain VKPM B-197 encodingIDO(Lys,32) is shown as SEQ ID NO: 7.

The following is an explanation of the method for obtaining the aminoacid sequence of IDO from IDO-producing bacteria.

The major protein was recovered from a gel and identified by MS-analysisas a putative RBTH_(—)06809 protein from Bacillus thuringiensis (serovarisraelensis) ATCC 35646 strain (FIG. 13).

Mass spectrometry analysis of the protein sample extracted from SDS-PAGEwas conducted. The treatment of gels, trypsinolysis, protein extraction,and mass analysis by time-of-flight matrix-assisted laserdesorption-ionization (MALDI-TOF) were conducted according to theprotocols described by Govorun, V. M. et al (The proteome comparativeanalysis of Helicobacter pylori clinical isolates. Biochemistry (Rus),68, 42-49 (2003)). The protein was identified by the set of itsphotolytic peptide masses using the Peptide Fingerprint option of Mascotsoftware (Matrix Science, USA). The DNA fragment coding for IDO can beobtained by PCR using appropriate primers designed based on the sequencefrom Bacillus thuringiensis (serovar israelensis) (ATCC 35646).

The procedure employed for PCR is described in publications such asWhite, T. J. et al., Trends Genet. 5, 185 (1989). The methods forisolating the chromosomal DNA, as well as the methods for isolating adesired DNA molecule from a gene library using a DNA molecule as aprobe, are described in publications such as Molecular Cloning, 3rdedition, Cold Spring Harbor Laboratory Press (2001).

A method for determining the nucleotide sequence of the isolated DNAthat encodes IDO is described in A Practical Guide to Molecular Cloning,John Wiley & Sons, Inc. (1985). Furthermore, the nucleotide sequence maybe determined by using the DNA Sequencer made by Applied Biosystems. TheDNA encoding IDO(Lys,23) derived from Bacillus thuringiensis strain2-e-2 is shown in SEQ ID No: 1. The nucleotide sequence from Bacillusthuringiensis (serovar israelensis) strain VKPM B-197 that encodesIDO(Lys,32) is shown in SEQ ID No: 7.

The DNA that encodes IDO is not only the DNA shown in SEQ ID No: 1, butalso includes functional variants of this sequence. This is becausethere are often differences in nucleotide sequences of the same functionamong various species and strains within the Bacillus genera. Suchfunctional variants will still encode an IDO protein which catalyzes theproduction of (2S,3R,4S)-4HIL from L-isoleucine.

The DNA encoding IDO may also include mutations which have beenartificially introduced as long as the mutated DNA still encodes IDOthat has the activity of catalyzing the production of (2S,3R,4S)-4HILfrom L-isoleucine. Methods for artificially adding mutations includecommonly known methods such as by introducing site-specific mutations asdescribed in Method. in Enzymol., 154 (1987).

The DNA also includes DNA that hybridizes under stringent conditionswith a nucleotide sequence complementary to the nucleotide sequence ofSEQ ID No: 1, and encodes a protein having IDO activity. As used herein,the “stringent conditions” refer to conditions under which a specifichybrid is formed and a non-specific hybrid is not formed. Although it isdifficult to numerically explicitly express these conditions, by way ofexample, conditions under which DNA molecules having higher homologye.g. preferably 70% or more, more preferably 80% or more, still morepreferably 90% or more, and particularly preferably 95% or more,hybridize with each other, while DNA molecules having lower homology donot hybridize with each other. Stringent conditions can also be definedas when hybridization occurs with typical washing conditions in Southernhybridization, that is, at a salt concentration corresponding to 0.1×SSCand 0.1% SDS at 37° C., preferably 0.1×SSC and 0.1% SDS at 60° C., andmore preferably 0.1×SSC and 0.1% SDS at 65° C. The length of the probemay be suitably selected, depending on the hybridization conditions, andusually varies from 100 bp to 1 kbp. Furthermore, “L-isoleucinedioxygenase activity” means the activity that results in the synthesisof (2S,3R,4S)-4HIL from L-isoleucine. However, when a nucleotidesequence hybridizes under stringent conditions with a nucleotidesequence complementary to the nucleotide sequence of SEQ ID No: 1,L-isoleucine dioxygenase activity is retained at least 10% or more,preferably 30% or more, more preferably 50% or more, and still morepreferably 70% or more, of the protein having the amino acid sequence ofSEQ ID No: 2, at 37° C. and pH 8.

Furthermore, the DNA may include a DNA encoding a protein which issubstantially identical to the IDO encoded by the DNA of SEQ ID No: 1.Namely, this may include the following DNAs:

(a) a DNA of the nucleotide sequence of SEQ ID No: 1;

(b) a DNA that hybridizes under stringent conditions with a DNA having anucleotide sequence complementary to the nucleotide sequence of SEQ IDNo: 1, and encodes a protein having L-isoleucine dioxygenase activity;

(c) a DNA that encodes a protein of the amino acid sequence of SEQ IDNo: 2;

(d) a DNA that encodes a protein having the amino acid sequence of SEQID NO: 2, but which includes substitutions, deletions, insertions,additions, or inversions of one or several amino acids, and hasL-isoleucine dioxygenase activity; and

(e) a DNA that encodes a protein having an amino acid sequence that isat least 70% homologous, preferably at least 80% homologous, morepreferably at least 90% homologous, and still more preferably at least95% homologous to the amino acid sequence of SEQ ID NO:2, and which hasthe L-isoleucine dioxygenase activity.

Here, “one or several” refers to the range over which the 3D structureof the protein having L-isoleucine dioxygenase activity is notsignificantly impaired, and more specifically, a range of 1 to 78,preferably 1 to 52, more preferably 1 to 26, and still more preferably 1to 13.

The substitution, deletion, insertion, addition, or inversion of one orseveral amino acid residues should be conservative mutation(s) so thatthe activity is maintained. The representative conservative mutation isa conservative substitution. Examples of conservative substitutionsinclude substitution of Ser or Thr for Ala, substitution of Gln, His orLys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn,substitution of Asn, Glu or Gln for Asp, substitution of Ser or Ala forCys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gln,substitution of Asn, Gln, Lys or Asp for Glu, substitution of Pro forGly, substitution of Asn, Lys, Gln, Arg or Tyr for His, substitution ofLeu, Met, Val or Phe for Ile, substitution of Ile, Met, Val or Phe forLeu, substitution of Asn, Glu, Gln, His or Arg for Lys, substitution ofIle, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, Ile or Leufor Phe, substitution of Thr or Ala for Ser, substitution of Ser or Alafor Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe orTrp for Tyr, and substitution of Met, Ile or Leu for Val.

Furthermore, “L-isoleucine dioxygenase activity” refers to the activitythat results in the synthesis of (2S,3R,4S)-4HIL from L-isoleucine asdescribed above. However, when the amino acid sequence contains one ormore substitutions, deletions, insertions, additions, or inversions ofone or several amino acid residues, the protein should retain at least10% or more, preferably 30% or more, more preferably 50% or more, andstill more preferably 70% or more L-isoleucine dioxygenase activity ascompared to the protein having the exact amino acid sequence of SEQ IDNo: 2, at 30° C. and pH 6.0. The L-isoleucine dioxygenase activity ofthe IDO can be measured by analysis of (2S,3R,4S)-4HIL formation fromL-isoleucine using high-performance liquid chromatography (HPLC).

Furthermore, a homologue DNA of SEQ ID NO: 1 can also be used. Whetherthe homologue DNA encodes L-isoleucine dioxygenase or not can beconfirmed by measuring L-isoleucine dioxygenase activity of the celllysate, or cell lysate of the microorganism in which the homologue DNAis overexpressed.

The homologue DNA of SEQ ID NO: 1 can also be prepared from the genomeof another Bacillus species, for example, Bacillus cereus or Bacillusweihenstephanensis.

The alignment of the amino acid sequences of Bacillus cereus, Bacillusthuringiensis, Bacillus weihenstephanensis is shown in FIG. 20, and theconserved sequence among the species of the genus Bacillus is shown inSEQ ID NO: 6.

Furthermore, DNA homologues of genes encoding IDO from other species andgenus of bacteria can be obtained by cloning, based on homologies to thefollowing-listed genes (Table.1) from Bacillus, Escherichia,Corynebacterium, Arthrobacter, Aspergillus, Pseudomonas, Granulibacter,Methylobacillus, Granulibacter, Acidiphilium, Agrobacterium,Gluconobacter, Caulobacter, Stigmatella, Myxococcus, Polaromonas,Caulobacter, Polaromonas, Sphingomonas, Acidovorax, Mycobacterium,Azotobacter, Vibrio, Polynucleobacter, Streptomyces, or the like. Thehomologues may be amplified by PCR using, for example, the syntheticoligonucleotides shown in SEQ ID NOS: 3 and 4.

TABLE 1 List of putative DNA encoding L-isoleucine dioxygenase GenbankAccession Gene Microorganism Description No. RBTH_06809 Bacillusthuringiensis Hypothetical protein AAJM01000012.1 serovar israelensisATCC RBTH_06809 GI: 75758796 35646 BC1061 Bacillus cereus ATCChypothetical protein NC_004722.1 14579 GI: 30019216 — Bacillus conservedZP_01182590.1| weihenstephanensis hypothetical protein GI: 89204011KBAB4 PSPPH_3986 Pseudomonas syringae pv. hypothetical proteinNC_005773.3 phaseolicola 1448A GI: 71735316 GbCGDNIH1 Granulibacterbethesdensis hypothetical protein NC_008343.1 2096 CGDNIH1 GI: 114328760Mfla_2629 Methylobacillus flagellatus hypothetical protein NC_007947.1KT GI: 91776977 GbCGDNIH1_2096 Granulibacter bethesdensis hypotheticalprotein NC_008343.1 CGDNIH1 GI: 114328760 — Acidiphilium cryptum JF-5conserved ZP_01144511.1 hypothetical protein GI: |88939060 —Agrobacterium vitis hypothetical protein ABG82019.1 GI: 110671820GOX1674 Gluconobacter oxydans hypothetical protein YP: 192070.1 621H GI:58040106 — Caulobacter sp. K31 conserved ZP: 01420729.1 hypotheticalprotein GI: 113934829 — Stigmatella aurantiaca conserved ZP_01462001.1DW4/3-1 hypothetical protein GI: 115374724 MXAN_6813 Myxococcus xanthusDK hypothetical protein YP: 634930. 1622 GI: 108759113 Bpro_0594Polaromonas sp. JS666 hypothetical protein YP: 547452.1 GI: 91786500CC3057 Caulobacter crescentus hypothetical protein NP_421851.1 CB1 GI:16127287 — Polaromonas similar to ZP_01022090.1 naphthalenivorans CJ2Uncharacterized GI: 84714798 protein conserved in bacteria —Sphingomonas sp. SKA58 putative phage ZP_01302473 repressor GI: 94495894Acidovorax sp. JS42 conserved ZP_01384166.1 hypothetical protein GI:110595841 Mycobacterium sp. JLS conserved ZP_01276363.1 hypotheticalprotein GI: 92907583 Azotobacter vinelandii similar to ZP_00417642.1AvOP Uncharacterized GI 67156016 protein conserved in bacteria VV21380Vibrio vulnificus CMCP6 hypothetical protein NP_763273.1 GI 27367746VVA0217 Vibrio vulnificus YJ016 hypothetical protein NP_936273.1 GI37675877 — Polynucleobacter sp. conserved ZP_01493168.1 QLW-P1DMWA-1hypothetical protein GI 116268923 AF484556_24 Streptomyces atroolivaceusconserved AAN85502.1 hypothetical protein GI: 26541515

(2) Properties of IDO

Next, an explanation is provided of the properties of purifiedL-isoleucine dioxygenase derived from Bacillus thuringiensis strain2-e-2 (IDO(Lys,23)).

The IDO(Lys,23) has the amino acid sequence of SEQ ID No: 2 as wasclearly determined by the previously described gene isolation andanalysis. However, other proteins may also have the IDO activity, butthe amino acid sequence may contain one or several substitutions,deletions, insertions, additions, or inversions of amino acid(s) ascompared to the amino acid sequences shown in SEQ ID No: 2, 8, 13, 17,or 21.

Namely, the IDO includes the following proteins:

(f) a protein having the amino acid sequences of SEQ ID No: 2, 8, 13,17, or 21;

(g) a protein having the amino acid sequences of SEQ ID No: 2, 8, 13,17, or 21, but which contains one or more substitutions, deletions,insertions, additions, or inversions of amino acid(s), and hasL-isoleucine dioxygenase activity; and

(h) a protein that is at least 70% homologous, preferably at least 80%homologous, more preferably at least 90% homologous and still morepreferably at least 95% homologous to the amino acid sequence of SEQ IDNO: 2, 8, 13, 17, or 21 and has L-isoleucine dioxygenase activity.

Here, the definitions of “several” and “L-isoleucine dioxygenaseactivity” are the same as defined in section (1), DNA EncodingL-isoleucine dioxygenase.

The IDO catalyzes the reaction that results in synthesis of(2S,3R,4S)-4HIL by hydroxylation from L-isoleucine.

The L-isoleucine dioxygenase activity of the IDO may be measured byanalysis of (2S,3R,4S)-4HIL formation from L-isoleucine usinghigh-performance liquid chromatography (HPLC).

The IDO is able to catalyze the reaction that results in synthesis of(2S,3R,4S)-4HIL by hydroxylation from L-isoleucine. In the hydroxylationreaction catalyzed by dioxygenases, one atom of molecular oxygen isincorporated into L-isoleucine, while the other oxygen atom isincorporated in another oxygen acceptor, for example, α-ketoglutarate,resulting in the formation of (2S,3R,4S)-4HIL and succinate with therelease of carbon dioxide. Dioxygenases are capable of hydroxylating analiphatic carbon chain in a stereospecific way. One plant enzyme hasbeen reported which is capable of catalyzing a hydroxylation reaction ofL-isoleucine, and that is the L-isoleucine dioxygenase derived fromfenugreek extract (Phytochemistry, Vol. 44, No. 4, pp. 563-566, 1997).However, this method is unsatisfactory for manufacturing4-hydroxy-L-isoleucine because the activity of the enzyme is inhibitedby isoleucine at concentrations of 20 mM and above, the enzyme has notbeen identified, the enzyme is derived from plant extracts and is notreadily obtained in large quantities, and the enzyme is unstable.

Next, the following provides a description of the enzymatic propertiesinvestigated for purified IDO(Lys,23).

IDO(Lys,23) catalyzes the reaction that forms (2S,3R,4S)-4HILrepresented by the following general formula (I):

from L-isoleucine in the reaction shown below:

L-isoleucine+α-ketoglutarate+O₂→4HIL+succinate+CO₂

Thus, a process of producing (2S,3R,4S)-4HIL from L-isoleucine by usingIDO(Lys,23) is also described.

Furthermore, the activity of IDO(Lys,23) strictly depends on thebivalent cation Fe²⁺ and is completely blocked in the presence of EDTA.IDO(Lys,23) is able to catalyze the transfer of one oxygen atom toL-isoleucine and another oxygen atom to α-ketoglutarate. So, IDO(Lys,23)may be an α-ketoglutamate-dependent dioxygenase.

The molecular weight of each IDO (Lys,23) subunit as measured bySDS-PAGE is about 29±2.0 kDa. Therefore, IDO includes proteins definedby the following characteristics:

(A) an activity that catalyzes production of (2S,3R,4S)-4HIL fromL-isoleucine and α-ketoglutarate;

(B) the activity is dependent on a bivalent cation such as Fe²⁺, and

(C) the molecular weight per subunit as measured by SDS-PAGE is about29±2.0 kDa.

The IDO(Lys,32) from Bacillus thuringiensis (serovar israelensis) strainVKPM B-197 has the amino acid sequence of SEQ ID NO: 8.

(3) Process for Producing L-isoleucine Dioxygenase

Next, an explanation is provided for the process of producing the IDO.There are two ways to produce IDO. These are (i) cultivating anIDO-producing microorganism to produce and accumulate IDO, and (ii)preparing a transformant to produce IDO by a recombinant DNA technologyand cultivating the transformant to produce IDO.

(i) Process for Forming and Producing IDO by Microbial Cultivation

Examples of microorganisms which can produce IDO by cultivation includemicroorganisms belonging to the genus Escherichia, Pseudomonas,Corynebacterium, Arthrobacter, Aspergillus, or Bacillus.

Any microorganism belonging to the genus Bacillus, Escherichia,Corynebacterium, Arthrobacter, Aspergillus, Pseudomonas, Granulibacter,Methylobacillus, Granulibacter, Acidiphilium, Agrobacterium,Gluconobacter, Caulobacter, Stigmatella, Myxococcus, Polaromonas,Caulobacter, Polaromonas, Sphingomonas, Acidovorax, Mycobacterium,Azotobacter, Vibrio, Polynucleobacter, Streptomyces may be used providedthey are microorganisms that produce the IDO which catalyzes thesynthesis of (2S,3R,4S)-4HIL from L-isoleucine and α-ketoglutarate.Preferable microorganisms include Bacillus thuringiensis strain 2-e-2and Bacillus thuringiensis (serovar israelensis; ATCC 35646) strain.Among these, Bacillus thuringiensis strain 2-e-2 is particularlypreferable.

Although the microorganism may be cultivated by any method, such asliquid or solid cultivation, an industrially advantageous method is byconducting deep-aerated stir cultivation. Carbon sources, nitrogensources, inorganic salts, and other trace nutrient elements commonlyused in microbial cultivation may be used. The chosen nutrient sourcesshould be appropriate for the chosen microorganism.

Culturing is conducted under aerobic conditions by shake-culturing, deepventilation stir-culturing, or the like. The cultivation temperature maybe within a range in which the microorganisms will grow and IDO isproduced. Thus, although the conditions are not strictly set forth, thecultivating temperature is typically 10 to 50° C. and preferably 15 to42° C. The cultivating time varies according to the other cultivatingconditions. For example, the microorganisms may be cultivated until thelargest amount of IDO is produced, and this is typically about 5 hoursto 7 days, and preferably about 10 hours to 96 hours.

Following cultivation, the microbial cells are recovered bycentrifugation (e.g., 10,000×g for 10 minutes). Since the majority ofthe IDO is present in the cells, the IDO is solubilized by disrupting orlysing the microbial cells. Disruption of the cells may be accomplishedby ultrasonic means, a French press, or glass beads. When lysing thecells, an egg white lysozyme, peptidase treatment, or a suitablecombination of these may be used.

When IDO is purified from the microorganism using an enzyme solubilizingsolution and undisrupted or unlysed residue remains, re-centrifuging thesolubilization solution and removing any residue that precipitates isadvantageous to purification.

All commonly used methods for purifying ordinary enzymes may be employedto purify the IDO, examples of which include ammonium sulfateprecipitation, gel filtration chromatography, ion exchangechromatography, hydrophobic interaction chromatography andhydroxyapatite chromatography. As a result, an IDO-containing fractionwith higher specific activity may be obtained.

(ii) Production Process Using Recombinant DNA Technology

Next, a process for producing IDO using recombinant DNA technology isdescribed. There are numerous known examples of producing usefulproteins such as enzymes and physiologically active substances usingrecombinant DNA technology. The use of recombinant DNA technologyenables mass production of useful proteins which are present in traceamounts in nature.

FIG. 11 is a flowchart of the process for producing the IDO.

First, a DNA is prepared that encodes IDO (Step S1).

Next, the prepared DNA is ligated with a vector DNA to produce arecombinant DNA (Step S2), and cells are transformed with therecombinant DNA to produce a transformant (Step S3). Then, thetransformant is cultivated in a medium, and the IDO is produced andaccumulates in the medium, the cells, or both (Step S4).

Subsequently, the process proceeds to Step S5 where IDO is recovered andpurified.

The desired (2S,3R,4S)-4HIL may be produced in a large amount by usingthe purified IDO produced at Step S5, or the medium, cells, or both inwhich IDO has accumulated from Step S4 in the hydroxylation reaction(Step S6).

The DNA that is ligated with the vector DNA may allow expression of theIDO.

Here, examples of IDO genes ligated into the vector DNA include thepreviously described DNA as described in [I].

For large scale protein production using recombinant DNA technology,host cells such as bacterial cells, Actinomyces cells, yeast cells, moldcells, plant cells, and animal cells may be transformed. Examples ofbacterial cells for which host-vector systems have been developedinclude Escherichia, Pseudomonas, Corynebacterium, Arthrobacter,Aspergillus, and Bacillus, and preferably Escherichia coli orCorynebacterium glutamicum is used. This is because there is a largevolume of knowledge regarding technologies for mass production ofprotein using Escherichia coli, Corynebacterium glutamicum, and Bacillusbacteria. The following describes a process for producing L-isoleucinedioxygenase using transformed E. coli. The following method for E. colican also be applied to Corynebacterium glutamicum and Bacillus bacteria.

Promoters typically employed in heterogeneous protein production in E.coli may be chosen to express the DNA encoding IDO, examples of whichinclude known strong promoters such as T7 promoter, trp promoter, lacpromoter, tac promoter, and PL promoter.

In order to produce IDO as a fused protein inclusion body, a gene thatencodes another protein, preferably a hydrophilic peptide, is ligatedeither upstream or downstream of the IDO gene. The gene that encodes theother protein may be a gene that increases the amount of fused proteinwhich accumulates and thereby enhances the solubility of the fusedprotein following the denaturation and regeneration steps. Examples ofcandidates include the T7 gene 10, β-galactosidase gene, dehydrofolatereductase gene, interferon-γ gene, interleukin-2 gene, and prochymosingene.

When ligating these genes with a gene that encodes IDO, the codonreading frames should be placed in frame. The genes may either beligated into a suitable restriction enzyme site or synthetic DNA of anappropriate sequence may be used.

In order to increase the amount produced, it is preferable to couple atranscription termination sequence downstream from the fused proteingene. Examples of such a transcription terminator sequence include theT7 terminator, fd phage terminator, T4 terminator, tetracyclineresistance gene terminator, and E. coli trpA gene terminator.

Multi-copy vectors are preferable for introducing the gene that encodesIDO or a fused protein of IDO into E. coli, examples of which includeplasmids having a replication starting point derived from Col E1 such aspUC plasmids, pBR322 plasmids, or their derivatives. A “derivative”refers to a plasmid in which base substitution, deletion, insertion,addition, or inversion has occurred. Such changes may be caused by amutagen, UV irradiation, or by spontaneous or random mutation.

The vector may have a marker such as the ampicillin resistance gene sothat transformants may be selected. Examples of such vectors includecommercially available expression vectors which include a powerfulpromoter (such as pUC (Takara), pPROK (Clontech), and pKK233-2(Clontech)).

Recombinant DNA may be obtained by ligating a DNA fragment containing apromoter, a gene encoding IDO or a fused protein of IDO and anotherprotein, and a terminator are ligated in that order, with a vector DNA.

When E. coli is transformed using the recombinant DNA and thencultivated, IDO or the fused protein is expressed and produced. Strainsthat are typically used to express heterogeneous genes may be used asthe transformed host, and E. coli strain JM109 (DE3) and E. coli strainJM109 are particularly preferable. The transformation method and methodfor selecting the transformant are described in, for example, MolecularCloning, 3rd edition, Cold Spring Harbor Laboratory Press (2001).

When expressing the fused protein, the IDO may be cut out using arestricting protease such as blood coagulation factor Xa or kallikreinthat recognizes sequences which are not present in IDO.

A medium which is typically used to cultivate E. coli may be used,examples of which include M9-casamino acid medium and LB medium.Furthermore, the cultivation and production conditions may beappropriately selected according to the type of the marker and promoteron the chosen vector, and the chosen host microorganism.

The following method may be used to recover the IDO or fused protein. Ifthe IDO or fused protein is solubilized within microbial cells, then thedisrupted or lysed microbial cells may be used as a crude enzymesolution. In addition, the IDO or its fused protein may also be furtherpurified by precipitation, filtration, column chromatography, or othercommon techniques as necessary. An antibody may also be used to purifythe IDO or fused protein.

When a protein inclusion body is formed, it may be solubilized with adenaturant. Although the protein inclusion body may be solubilized withmicrobial cell protein, in consideration of the subsequent purificationprocedure, it is preferable to remove the inclusion body and thensolubilize it. A known method may be used to recover the inclusion bodyfrom the microbial cell. For example, the inclusion body may berecovered by disrupting the microbial cell followed by centrifugation.Examples of denaturants that solubilize protein inclusion bodies includeguanidine hydrochloride (e.g., 6 M, pH 5-8) and urea (e.g., 8 M).

The protein inclusion body may be regenerated as an active protein byremoving these denaturants by dialysis, for example. Dialysis solutionssuch as Tris-HCl buffer or phosphate buffer may be used for dialysis,and the concentration may be from 20 mM to 0.5 M, and the pH may be frompH 5 to pH 8.

The protein concentration during the regeneration step is preferablymaintained at about 500 μg/ml or less. In order to prevent theregenerated IDO from self-crosslinking, the dialysis temperature ispreferably 5° C. or lower. Furthermore, restoration of activity may alsobe accomplished by removing the denaturant by dilution and/orultrafiltration in addition to the dialysis.

When the IDO gene is derived from bacteria belonging to the genusBacillus, the IDO may be expressed and produced in host bacteria ofEscherichia, Pseudomonas, Corynebacterium, Arthrobacter, Aspergillus, orBacillus.

The copy number of the gene may be increased by inserting the gene intoa multi-copy vector, followed by introducing the vector into amicroorganism. Multi-copy vectors include E. coli plasmid vectors suchas pMW118, pBR322, pUC19, pBluescript KS+, pACYC177, pACYC184, pAYC32,pMW119, pET22b, E. coli-B. subtilis shuttle vectors such as pHY300PLK,pGK12, pLF14, pLF22 or the like, phage vectors such as 11059, IBF101,M13mp 9, Mu phage (Japanese Patent Application Laid-Open No. 2-109985),or the like, and transposons (Berg, D. E. and Berg, C. M., Bio/Technol.,1, 417 (1983)), such as Mu, Tn10, Tn5, or the like. It is also possibleto increase the copy number of a gene by integrating the gene into achromosome by homologous recombination utilizing a plasmid, or the like.Examples of host cells and expression systems useful for such purpose inArthrobacter sp. are described in Shaw P. C. et al. (J Gen Micobiol. 134(1988) p. 903-911) and Morikawa, M. et al. (Appl Microbiol Biotechnol.,42 (1994), p. 300-303). In Arthrobacter nicotinovorans, such systems aredescribed in Sandu C. et al. (Appl Environ Microbiol. 71 (2005) p8920-8924). Also, expression systems developed for Coryneform bacteriahave been reported to be functional in Arthrobacter species (Sandu C. etal.). However, the Bacillus species bacteria are not limited to thoserecited herein.

[II] Method for Producing (2S,3R,4S)-4-hydroxy-L-isoleucine

The method for producing (2S,3R,4S)-4-hydroxy-L-isoleucine((2S,3R,4S)-4HIL) represented by the general formula (I) includes a onestep reaction of direct enzymatic hydroxylation of L-isoleucine shownbelow:

L-isoleucine+α-ketoglutarate+O₂→4HIL+succinate+CO₂

wherein the reaction is performed in the presence of IDO, L-isoleucine,α-ketoglutarate, and one oxygen molecule. The L-isoleucine andα-ketoglutarate act as acceptor molecules, each accepting one oxygenatom. IDO catalyzes the reaction.

In the present invention, “enzymatic hydroxylation” means ahydroxylation reaction which is carried out by an IDO enzyme.Particularly, a bacterial IDO is preferred.

There are no particular limitations on the IDO that catalyzes thereaction, and any protein may be used as long as the protein is capableof catalyzing the reaction hydroxylation of L-isoleucine in the presenceof α-ketoglutarate and oxygen.

A preferable example of IDO is described in section [1]. The IDO may bepresent in the reaction in any form, including contained within abacterium (including a culture, bacterial cells, or treated cells), apurified enzyme, or a crude enzyme, so long as it is able to catalyzethe reaction which produces (2S,3R,4S)-4HIL. When bacteria is used asthe source of IDO, both (1) bacteria which naturally produce IDO, suchas microorganisms belonging to the genus Bacillus, and (2) recombinantmicroorganisms which have been transformed with recombinant DNA asdescribed in section [1] may be cultivated to produce IDO.

Amino acid sequences of IDO include those depicted in SEQ ID NO: 2, SEQID NO:8, and Table 1.

L-isoleucine dioxygenase also includes a protein as defined by followingcharacteristics:

(A) an activity that catalyzes the reaction of producing (2S,3R,4S)-4HILfrom L-isoleucine and α-ketoglutarate;

(B) the activity is dependent on a bivalent cation including Fe²⁺, and

(C) a molecular weight per subunit as measured by SDS-PAGE of about29±2.0 kDa.

For example, when producing (2S,3R,4S)-4HIL using IDO-producing bacteriaor bacterial cells that have been transformed with a recombinant DNA,the substrate may be added directly to the culture media duringcultivation, or the bacterial cells or washed bacterial cells that havebeen separated from the culture may be used directly. Furthermore,treated bacterial cells that have been disrupted or lysed may also beused directly, or the IDO may be recovered from the treated bacterialcells and used as a crude enzyme solution, or the enzyme may be purifiedprior to conducting the reaction. Namely, as long as IDO activity ispresent, regardless of the form of the composition, it may be used orproduce (2S,3R,4S)-4HIL.

In order to perform an hydroxylation reaction using IDO, a reactionsolution containing L-isoleucine, α-ketoglutarate, and IDO or anIDO-containing composition is adjusted to the suitable temperature of 20to 50° C., and either allowed to stand undisturbed, or agitated byshaking or stirring for 30 minutes to 5 days, while maintaining at pH 5to 12.

The reaction velocity may also be increased by adding a bivalent cationsuch as Fe²⁺ to the reaction mixture.

When adding bivalent cations to the reaction solution, although any saltmay be used provided it does not hinder the reaction, FeSO₄, and soforth are preferable. The concentrations of these bivalent cations maybe determined by simple preliminary studies conducted by a person withordinary skill in the art. These bivalent cations may be added withinthe range of 0.01 mM to 50 mM, preferably 0.1 mM to 25 mM.

Oxygen enters the reaction from the air as a result of agitation of afixed culture volume during cultivation.

The (2S,3R,4S)-4HIL of general formula (I) that is formed in thereaction mixture may be either separated or purified according to knowntechniques, or further processed, particularly when a recombinantmicroorganism expresses IDO.

Examples of separation and purification methods include contacting the(2S,3R,4S)-4HIL with an ion exchange resin to adsorb basic amino acidsfollowed by elution and crystallization, and eluting the discoloredproduct, filtrating with activated charcoal, and finally conductingcrystallization.

Non-annotated genes encoding IDO from other microorganisms can beidentified by their homology to the known IDO genes, followed byevaluation of the activity of proteins encoded by these genes.

Homology between two amino acid sequences can be determined bywell-known methods, for example, the computer program BLAST 2.0, whichcalculates three parameters: score, identity, and similarity.

Therefore, the DNA fragment from Bacillus thuringiensis strain 2-e-2 andBacillus thuringiensis (serovar israelensis; ATCC 35646) strain encodingthe full-length IDO can be obtained by PCR (polymerase chain reaction;refer to White, T. J. et al., Trends Genet., 5, 185 (1989)) utilizingprimers prepared based on the known amino acid and nucleotide sequences.The DNA fragment encoding IDO from other microorganisms can be obtainedin a similar manner.

Since there may be some differences in DNA sequences among the bacterialstrains, the above-described fragments encoding IDO are not limited tothe nucleotide sequences shown in SEQ ID NO: 1, 7, 12, 16, 20, FIG. 18or Table 1, but may also include nucleotide sequences similar to thoseshown in SEQ ID NO: 1, 7, 12, 16, 20, FIG. 18 or Table 1. Therefore, theprotein variants encoded by the above-described genes may have asimilarity of not less than 80%, preferably not less than 90%, and mostpreferably not less than 95%, with respect to the entire amino acidsequences shown in SEQ ID NOS. 2, 8, 13, 17, 21, FIG. 19 or Table 1, aslong as the abilities of the proteins to catalyze the desired reactionsare maintained.

Moreover, the above-described DNA fragments may be variants which canhybridize under stringent conditions with the nucleotide sequences shownin SEQ ID NOS: 1, 7, 12, 16, 20, FIG. 18 or Table 1, or with probesprepared based on these nucleotide sequences, provided that they encodefunctional proteins. “Stringent conditions” means the same as describedin section [1] (1) A DNA Encoding IDO.

The treated bacterial cells which may be employed include driedbacterial mass, freeze-dried bacterial mass, products treated withsurfactants or organic solvents, enzyme-treated products,ultrasound-treated products, mechanically ground products,solvent-treated products, protein fractions of bacterial mass,immobilized products of bacterial mass, and processed bacterial mass.

IDO may be prepared separately as described above, and then added to thereaction solution. A bacterium (host cell) that expresses the DNAencoding IDO may be prepared by transfecting the host cell with theexpression vector containing the DNA encoding the IDO so that is able tobe expressed in the chosen host cell. Furthermore, the host cells inwhich the expression of IDO has been increased, resulting in enhancedIDO activity, are preferably used.

The phrase “increasing the expression of the gene” means that theexpression of the gene is higher than that of a non-modified strain, forexample, a wild-type strain. Examples of such modifications includeincreasing the copy number of expressed gene(s) per cell, increasing theexpression level of the gene(s), and so forth. The quantity of the copynumber of an expressed gene is measured, for example, by restricting thechromosomal DNA followed by Southern blotting using a probe based on thegene sequence, fluorescence in situ hybridization (FISH), and the like.The level of gene expression can be measured by various known methodsincluding Northern blotting, quantitative RT-PCR, and the like. Theamount of the protein encoded by the gene can be measured by knownmethods including SDS-PAGE followed by immunoblotting assay (Westernblotting analysis), and the like.

“Transformation of a bacterium with DNA encoding a protein” means theintroduction of the DNA into the bacterium, for example, by conventionalmethods. Transformation of this DNA will result in an increase inexpression of the gene encoding the protein, and will enhance theactivity of the protein in the bacterial cells. Methods oftransformation include any known methods that have previously beenreported. For example, treating recipient cells with calcium chloride soas to increase permeability of the cells to DNA has been reported forEscherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159(1970)), and may be used.

Methods of enhancing gene expression include increasing the gene copynumber. Introducing a gene into a vector that is able to function in thechosen bacterium will increase the copy number of the gene. For suchpurposes, multi-copy vectors are preferably used, and include, forexample, pBR322, pMW119, pUC19, pET22b, or the like.

Gene expression may also be enhanced by introducing multiple copies ofthe gene into a bacterial chromosome by, for example, homologousrecombination, Mu integration, or the like. For example, one act of Muintegration allows for introduction of up to 3 copies of the gene into abacterial chromosome.

Increasing the copy number of the gene can also be achieved byintroducing multiple copies of the gene into the chromosomal DNA of thebacterium. In order to introduce multiple copies of the gene into abacterial chromosome, homologous recombination is carried out using atarget sequence which is present in multiple copies on the chromosomalDNA. Such sequences include, but are not limited to repetitive DNA, orinverted repeats, present at the end of a transposable element. Also, asdisclosed in U.S. Pat. No. 5,595,889, it is possible to incorporate thegene into a transposon, and transfer it so that multiple copies of thegene are introduced into the chromosomal DNA.

Enhancing gene expression may also be achieved by placing the DNA underthe control of a potent promoter. For example, the Ptac promoter, thelac promoter, the trp promoter, the trc promoter, the P_(R), or theP_(L) promoter of lambda phage are all known to be potent promoters. Theuse of a potent promoter can be combined with increasing the number ofgene copies.

Alternatively, the effect of the promoter can be enhanced by, forexample, introducing a mutation into the promoter to increase thetranscription level of a gene located downstream of the promoter.Furthermore, it is known that substitution of several nucleotides in thespacer region between the ribosome binding site (RBS) and the startcodon, especially the sequences immediately upstream of the start codon,profoundly affect the mRNA translation. For example, a 20-fold range inthe expression levels was found, depending on the nature of the threenucleotides preceding the start codon (Gold et al., Annu. Rev.Microbiol., 35, 365-403, 1981; Hui et al., EMBO J., 3, 623-629, 1984).Previously, it was shown that the rhtA23 mutation is an A-for-Gsubstitution at the −1 position relative to the ATG start codon(ABSTRACTS of 17th International Congress of Biochemistry and MolecularBiology in conjugation with 1997 Annual Meeting of the American Societyfor Biochemistry and Molecular Biology, San Francisco, Calif. Aug.24-29, 1997, abstract No. 457).

Moreover, it is also possible to introduce a nucleotide substitutioninto the promoter region of the gene on the bacterial chromosome, whichresults in stronger promoter function. The expression control sequencecan be altered, for example, in the same manner as the gene substitutionusing a temperature-sensitive plasmid, as disclosed in InternationalPublication WO 00/18935 and Japanese Patent Application Laid-Open No.1-215280.

Methods for preparing plasmid DNA include, but are not limited todigestion and ligation of DNA, transformation, selection of anoligonucleotide as a primer, and the like, or other methods well knownto one skilled in the art. These methods are described, for instance, in“Molecular Cloning A Laboratory Manual, Third Edition”, Cold SpringHarbor Laboratory Press (2001).

EXAMPLES

The present invention will be explained in further detail with referenceto the following non-limiting examples.

Example 1 Screening for Strains Having L-Ile Hydroxylase

<1> Screening for Strains able to Produce 4-Hydroxyisoleucine, andAnalysis of the Broth

By using L-isoleucine as a substrate, microorganisms were screened forthe presence of 4-hydroxyisoleucine. In water, 0.4% (w/v) of solublestarch, 0.4% of yeast extract, 1% of malt extract, and 0.2% ofL-isoleucine were dissolved, then the solution was adjusted to pH 7 to7.5. Soil bacteria were inoculated into this solution. After culturingwith shaking at 28° C. for 2 days, the presence of 4-hydroxyisoleucinewas determined by amino acid analysis of the centrifugation supernatant.

Amino Acid Analysis Conditions

4-Hydroxyisoleucine was detected by using the Waters AccQ-Tag™ method.Amino acids in 5 μl were diluted to an appropriate concentration andderivatized in a conventional manner, and the amount of4-hydroxyisoleucine was measured by HPLC analysis. As a result, one ofthe bacterial strains (strain 2-e-2) was found to have the activity ofproducing a substance showing the same retention time as that of4-hydroxyisoleucine. On the basis of the analysis of 16S rDNA, thestrain 2-e-2 was identified as Bacillus thuringiensis. Therefore, otherBacillus bacteria were also screened, and a similar activity was alsofound in Bacillus licheniformis (AKU 223, IAM 11054), Bacillussphaericus (AKU 227, NBRC 3526), and Bacillus thuringiensis (AKU 238,NBRC 3958).

Identification of Product

The substance produced from L-isoleucine as a substrate by the Bacillusstrains obtained in the aforementioned screening was identified. First,the molecular weight of the product was analyzed by MS, and found to be145, which is twice as small as that of 4-hydroxyisoleucine. When thecomposition formula was estimated by precise mass measurement using ahigh resolution mass spectrometer (Q-TofMS), C₆H₁₁NO₃ was obtained, andfound to have 2 times fewer hydrogen atoms than that of4-hydroxyisoleucine. The above results suggested that the substanceproduced by the Bacillus strains may be 2-amino-3-keto-4-methylpentanoicacid (AMKP). According to the experimental method previously described,AMKP was synthesized and purified from the culture media of Bacillusbacteria, and NMR analysis was performed (Bioorganic Chemistry, Vol. 6,pp. 263-271, 1977). As a result, both showed similar chemical shifts.

From the above, it was determined that the Bacillus bacteria found inthis experiment produced AMKP. The amount of AMKP produced by the strain2-e-2 was about 1 to 2 mM. The amount of AMKP produced by the Bacillusbacteria described in the Bioorganic Chemistry article 7 was 0.04 mM,and thus, the 2-e-2 strain was capable of producing a much larger amountthan previously described.

<2> Establishing a Method for the Separation and Analysis of AMKP andHIL

Since Bacillus bacteria were found to produce AMKP, a method for theseparation and analysis of AMKP and 4-hydroxyisoleucine was necessary.As a result of various examinations, the method for the separation andanalysis of AMKP and 4-hydroxyisoleucine was established by modifyingthe Waters AccQ-Tag™ method. Specifically, the column was changed toXBridge C18 5 mm, 2.1×150 mm (Waters), Eluent B was changed to MeOH, andthe flow rate of the eluent was changed to 0.3 ml/min. The gradient ofthe eluent is shown in the following table.

TABLE 2 Eluent conditions in a simultaneous analysis of HIL and AMKPAccq Tag 100% Time Flow Eluent A MeOH H₂O (min) (ml/min) % A % B % CCurve — 0.3 80 20 0 — 15   0.3 60 40 0  6 15.1 0.3 0 60 40 11 18.0 0.380 20 0 11

Under the above conditions, AMKP in the medium eluted at around 11.0minutes and 4-hydroxyisoleucine eluted at around 11.9 minutes. Thereforethese products could be separated.

<3> Change in Amount of AMKP Produced by the Addition of CofactorsDuring the Culture

The possibility of molecular oxygen uptake by hydroxylation wasconsidered as a possible mechanism for the production of AMKP.Accordingly, the production of AMKP was analyzed when NAD(P)H, or Fe²⁺,2-oxoglutaric acid and ascorbic acid, were added during the culture ofthe 2-e-2 strain. NAD(P)H is a cofactor of monooxygenases and Fe²⁺,2-oxoglutaric acid and ascorbic acid are cofactors of dioxygenases.

The culture medium used in the previous screening was used as a control,and the production of AMKP was compared among cultures containing thecofactors as shown in Table 3. The culture temperature was 30° C., andthe culture time was 22 hours. The concentration of AMKP in the culturesupernatants was measured. The results are shown in Table 3.

TABLE 3 Effect of addition of cofactors on AMKP production activity inculture of strain 2-e-2 AMKP concentration (mM) Control 1.34 NAD(P)H1.58 Fe²⁺ 2.53 Fe²⁺, 2-oxoglutaric 3.06 acid, ascorbic acid

It was suggested that a dioxygenase might be involved in the productionof AMKP.

<4> Changes in the 2-e-2 Strain Broth over Time

The AMKP production medium (0.4% (w/v) of soluble starch, 0.4% of yeastextract, 1% of malt extract, 0.2% of L-isoleucine, 0.5% of glucose, 1 mMascorbic acid, 1 mM 2-oxoglutaric acid, 1 mM CaCl₂, 1 mM MgSO₄, pH 7 to7.5) was put into a 3-L Sakaguchi flask, and the 2-e-2 strain wascultured at 23° C. with shaking. At 0, 6, 8, 10, 12, 14, 16, 18, and 20hours after the start of the culture, the culture broth was sampled, andthe 4-hydroxyisoleucine and AMKP were quantified by the method describedin <2> above. Further, turbidity (OD₆₆₀) was also measured.

The concentrations of 4-hydroxyisoleucine (HIL) and AMKP are shown inFIG. 1. The turbidity of the culture broth is shown in FIG. 2. The4-hydroxyisoleucine and AMKP increased in the logarithmic growth phaseand then reached a plateau. After the level reached the plateau,4-hydroxyisoleucine gradually decreased, and AMKP gradually increased.

At each of the aforementioned culture times, 2-e-2 cells were obtainedfrom 200 μl of the culture broth, washed with physiological saline, andthen suspended in 100 μl of a dioxygenase reaction mixture (10 mM Ile, 1mM Fe²⁺, 10 mM 2-oxoglutaric acid, 10 mM ascorbic acid, 50 mM potassiumphosphate buffer (pH 7.0)). The reaction occurred at 30° C. for 1 hourwith shaking, and the amounts of 4-hydroxyisoleucine and AMKP in thesupernatant were measured. As a result, only cells in the logarithmicgrowth phase were able to produce 4-hydroxyisoleucine and AMKP, althoughonly in trace amounts, as shown in FIG. 3.

<5> Production of HIL with 2-e-2 Cell Lysate

The cells were cultured in the AMKP medium until OD₆₆₀ of 3.2 wasachieved, then the collected and washed 2-e-2 cells were disrupted usinga mortar, and suspended in a buffer (50 mM HEPES (pH 7.0), 10% ofglycerol, Complete Mini (Roche)) until a suspension (lysate) is obtainedhaving a protein concentration of about 10 mg/ml includingcentrifugation precipitates. The centrifugation precipitates werefinally suspended in physiological saline of the same volume as the celllysate. These were each mixed with an equal volume of a 2× dioxygenasereaction mixture (10 mM Ile, 2 mM Fe²⁺, 10 mM 2-oxoglutaric acid, 10 mMascorbic acid, 100 mM HEPES (pH 7.0)), and the reaction proceeded at 30°C. for 1 hour. Similarly, resting cells used for the preparation of thecell lysate were washed, and suspended in physiological saline at aconcentration 10 times that in the culture broth, the suspension wasmixed with an equal volume of the 2× dioxygenase reaction mixture, andthe reaction proceeded. The amounts of AMKP and 4-hydroxyisoleucine inthe samples are shown in FIG. 4.

By using the cell lysate, for the production of 4-hydroxyisoleucineusing Ile as a substrate was confirmed.

<6> The Effects of Enzyme Cofactors in 2-e-2 Cell Lysate

By using a cell lysate of the 2-e-2 cells in the logarithmic growthphase, the effects of cofactors on the 4-hydroxyisoleucine productionreaction by hydroxylation of Ile were examined. A 50 mM HEPES (pH 7)reaction mixture was used containing final concentrations of 5 mM Ileand 5 mM of one of various cofactors with a cell lysate of the 2-e-2cells (lysate) in the logarithmic growth phase prepared by the methoddescribed in <5> above, and the amount of 4-hydroxyisoleucine producedwas measured. As shown in Table 4, Fe²⁺ (Fe) and 2-oxoglutaric acid(a-KG) were required for the production of 4-hydroxyisoleucine, and theamount of 4-hydroxyisoleucine which was produced was maximized by addingascorbic acid (Asc.). Therefore, such results strongly suggest that adioxygenase is involved in the production of 4-hydroxyisoleucine byhydroxylation of isoleucine.

TABLE 4 Effects of various cofactors on 4-hydroxyisoleucine productionactivity with 2-e-2 cell lysate Reaction mixture HIL production (mM)Lysate (—) 0.00 Lysate + Ile 0.00 Lysate + Ile + Fe 0.00 Lysate + Ile +Asc. 0.00 Lysate + Ile + a-KG 0.00 Lysate + Ile + Fe + Asc. 0.00Lysate + Ile + Fe + a-KG 0.39 Lysate + Ile + Asc. + a-KG 0.00 Lysate +Ile + Fe + Asc. + a-KG 0.85

<7> Steric Configuration of Product Obtained with 2-e-2 Cell Lysate

4-Hydroxyisoleucine has asymmetric carbons at 3 sites, and 8 types ofdiastereomers and 4 pairs of enantiomers exist. Specifically, the 4pairs of enantiomers are the following: the (2S,3S,4S) and (2R,3R,4R)enantiomers (also referred to as HIL1), the (2S,3S,4R) and (2R,3R,4S)enantiomers (also referred to as HIL2), the (2S,3R,4R) and (2R,3S,4S)enantiomers (also referred to as HIL3), and the (2S,3R,4S) and(2R,3S,4R) enantiomers (also referred to as HIL4). The naturallyoccurring HIL in Fenugreek and so forth is the (2S,3R,4S) enantiomer.Since (2S,3S)-isoleucine is used as a substrate, the 4-hydroxyisoleucineproduced by the hydroxylation reaction is either the (2S,3R,4S) or(2S,3R,4R) enantiomer. Accordingly, the steric configuration of4-hydroxyisoleucine produced by the 2-e-2 was determined.

(2R,3R,4R): HIL1 and (2S,3R,4R): HIL3 were obtained according toTetrahedron (47(32), 6469-6482, (1991)), and (2R,3R,4S): HIL2 and(2S,3R,4S): HIL4 were obtained according to Eur. J. Org. Chem. (834-839,(2002)). When HIL1 to HIL4 were analyzed under the conditions of thesimultaneous analysis of 4-hydroxyisoleucine and AMKP described in <2>above, the retention times were as shown in Table 5.

TABLE 5 Retention times of diastereomers of 4-hydroxyisoleucineRetention time (min) HIL1 7.25 HIL2 14.28 HIL3 11.10 HIL4 11.98

When the sample prepared in <6> was analyzed, the retention time was11.99 minutes. When it was mixed with 4-hydroxyisoleucine of thestandard of HIL4 and analyzed, the peaks completely matched. Therefore,when (2S,3S)-isoleucine was used as the substrate, the4-hydroxyisoleucine that was produced was HIL4, i.e., the naturallyoccurring 4-hydroxyisoleucine.

<8> Optimum pH of Enzyme in 2-e-2 Cell Lysate

By using a cell lysate prepared from 2-e-2 cells at OD₆₆₀ of 7 accordingto the method described in <5>, the pH dependency of the4-hydroxyisoleucine production was evaluated. The dioxygenase reactionmixture contained 5 mM Ile, 5 mM Fe²⁺, 5 mM 2-oxoglutaric acid, 5 mMascorbic acid, and 100 mM GTA, and the pH was measured after thereaction. The reaction temperature was 30° C. The 4-hydroxyisoleucinewas analyzed by the method described in <2>. Activities at various pHvalues are shown in FIG. 5 in terms of relative activity ratioscalculated on the basis of the amount of HIL produced at the pH whichresults in the maximum amount produced, which was taken as 100%. Theactivity was confirmed at pH 5 to 8.

<9> Optimum Temperature of Enzyme in the 2-e-2 Cell Lysate

By using a cell lysate prepared from 2-e-2 cells at OD₆₆₀ of 7 accordingto the method described in <5>, the temperature dependency of the4-hydroxyisoleucine production was evaluated. The dioxygenase reactionmixture contained 5 mM Ile, 5 mM Fe²⁺, 5 mM 2-oxoglutaric acid, 5 mMascorbic acid and 100 mM GTA (pH 6), and the reaction temperature was 15to 50° C. The 4-hydroxyisoleucine which was produced was analyzed by themethod described in <2>. Activities at various temperatures are shown inFIG. 6 in terms of relative activity ratios calculated on the basis ofthe amount of 4-hydroxyisoleucine produced at the temperature whichresults in the maximum amount produced, which was taken as 100%. Theoptimum temperature was lower than 45° C.

<10> Temperature Stability of Enzyme in 2-e-2 Cell Lysate

By using a cell lysate prepared from 2-e-2 cells at OD₆₆₀ of 7 accordingto the method described in <5>, the temperature stability of the4-hydroxyisoleucine production was evaluated. The cell lysate wasincubated at 0 to 50° C. for 1 hour, and then the Ile hydroxylationactivity was measured. The substrate reaction mixture contained 5 mMIle, 5 mM Fe²⁺, 5 mM 2-oxoglutaric acid, 5 mM ascorbic acid, and 100 mMHEPES (pH 7), and the reaction temperature was 30° C. The4-hydroxyisoleucine which was produced was analyzed by the methodmentioned in Example 2. Temperature stabilities at various temperaturesare shown in FIG. 7 in terms of relative activity ratios calculated onthe basis of the amount of 4-hydroxyisoleucine produced at thetemperature which results in the maximum amount produced, which wastaken as 100%. The enzyme was inactivated at a temperature of 50° C. orhigher.

<11> Substrate Reaction Characteristics of Enzyme in 2-e-2 Cell Lysate

By using a cell lysate prepared from 2-e-2 cells at OD₆₆₀ of 7 accordingto the method described in <5>, the reaction characteristics for variousamino acids were evaluated. The cell lysate and a substrate solutionwere mixed, then the reaction proceeded at 30° C. for 1 hour, and theproduction of new substances was evaluated by TLC or amino acidanalysis. The substrate reaction mixture contained 5 mM amino acid, 5 mMFe²⁺, 5 mM 2-oxoglutaric acid, 5 mM ascorbic acid, and 100 mM HEPES (pH7). In addition to L-isoleucine, the amino acids L-leucine, L-valine,L-glutamic acid, and L-lysine were each individually evaluated. The4-hydroxyisoleucine which was produced was analyzed by the methoddescribed in Example 1. The results are shown in Table 6. Production ofamino acids other than L-isoleucine was not observed. Therefore, it wassuggested that this enzyme was an isoleucine-specific dioxygenase.

TABLE 6 Reactivity for various amino acids Product L-Isoleucine Produced(HIL) L-Leucine None L-Valine None L-Glutamic acid None L-Lysine None

<12> Effect of Inhibitors in the 2-e-2 Cell Lysate

By using a cell lysate prepared from 2-e-2 cells at OD₆₆₀ of 7 accordingto the method described in <5>, the effects of inhibitors on the4-hydroxyisoleucine production were examined. A cell lysate preparedfrom 2-e-2 cells at OD₆₆₀ of 7 according to the method described in <5>was used. The dioxygenase reaction mixture contained 5 mM Ile, 5 mMFe²⁺, 5 mM 2-oxoglutaric acid, 5 mM ascorbic acid, and 100 mM HEPES (pH6), the reaction temperature was 30° C., and the reaction time was 1hour. The amount of 4-hydroxyisoleucine which was produced when 10 mM ofeach inhibitor (EDTA, Cu²⁺, Zn²⁺) was added to the reaction system wasmeasured. 4-Hydroxyisoleucine was analyzed by the method described in<2>. The isoleucine hydroxylation activity was inhibited by theinhibitors.

Example 2 Isolation and Purification of L-Ile Hydroxylase

(1) Preparation of Cell-Free Extract

The 2-e-2 strain was cultured in a total volume of 2 L of AMKPproduction medium until OD₆₆₀ of 6.0 was achieved, and then the cellswere washed with physiological saline. The cells were suspended in acell membrane treatment solution (10 mg/ml of lysing enzyme (SIGMA), 5mg/ml of cellulase “ONOZUKA” R-10 (Yakult), Yatalase (Takara Bio), 1mg/ml of lysozyme (SIGMA), dissolved in 0.2 M NaH₂PO₄ and 0.6 M KCl (pH5.5)), and then incubated at 30° C. for 1 hour. The incubated cells werewashed with physiological saline, and then suspended in Buffer A (50 mMHEPES (pH 7.0), 10% of glycerol, 2 mM DTT, 1 mM EDTA, Complete (Roche)),and the cells were disrupted by using an ultrasonic disruptor (Branson)while on ice. This treated suspension was centrifuged at 4° C. and18,500×g for 60 minutes to obtain a supernatant. The subsequentisolation and purification procedures were all performed at 4° C. or onice.

(2) Anion Exchange Chromatography

The supernatant obtained in the previous step was filtered through afilter with a pore size of 0.45 μm, and applied to a DEAE column (16mm×100 mm, GE Healthcare Bio-Sciences) equilibrated beforehand withBuffer A. The column was washed with Buffer A, and elution was performedwith a linear concentration gradient of sodium chloride in Buffer B (50mM HEPES (pH 7.0), 10% of glycerol, 2 mM DTT, 1 mM EDTA, 0.5 M NaCl,Complete (Roche)).

(3) Detection of Active Fraction

Each fraction was reacted using an Ile hydroxylation activity reactionmixture (100 mM HEPES (pH 6.0), 5 mM L-Ile, 5 mM Fe²⁺, 5 mM2-oxoglutaric acid, 5 mM ascorbic acid at final concentrations) at 30°C. for 30 minutes. The enzyme was inactivated at 100° C., and then theamount of 4-hydroxyisoleucine was quantified by the aforementioned AMKPand HIL separation and measurement method. The 4-hydroxyisoleucinereferred to in the subsequent analysis indicates a substance that issubstantially composed of the isomer having the same retention time asthat of the naturally occurring 4-hydroxyisoleucine. The enzyme activitythat produces 1 nmol of HIL per minute was defined as 1 U.

(4) Cation Exchange Chromatography

The buffer in the active fraction obtained in the previous step waschanged to Buffer C (50 mM MES (pH 5.2), 10% of glycerol, 2 mM DTT, 1 mMEDTA, Complete (Roche)) in a desalting column (GE HealthcareBio-Sciences). Then it was applied to a MonoS column (10 mm×100 mm, GEHealthcare Bio-Sciences) equilibrated beforehand with Buffer C. Thecolumn was washed with Buffer C, then eluted with a linear concentrationgradient of sodium chloride in Buffer D (50 mM MES (pH 5.2), 10% ofglycerol, 2 mM DTT, 1 mM EDTA, 0.5 M NaCl, Complete (Roche)), and theIle hydroxylation activity of each fraction was measured.

(5) Ammonium Sulfate Precipitation

2 M Ammonium sulfate was added to the soluble fraction obtained in theprevious step and dissolved. The solution was sufficiently stirred, andthen fractionated into a soluble fraction and a precipitate fraction bycentrifugation, and the precipitate fraction was dissolved in Buffer A.When the Ile hydroxylation activity of each fraction was measured, theactivity was detected in the precipitate fraction.

(6) Size Exclusion Chromatography

The active fraction obtained in the previous step was applied to aSuperdex 75 column (10 mm×300 mm, GE Healthcare Bio-Sciences)equilibrated beforehand with Buffer A. Elution was performed with BufferA, and the Ile hydroxylation activity of each fraction was measured.

(7) Hydrophobic Interaction Chromatography

The buffer in the active fraction obtained in the previous step waschanged to Buffer E (50 mM MES (pH 6.5), 10% of glycerol, 2 mM DTT, 1 mMEDTA, 1 M ammonium sulfate, Complete (Roche)). The this fraction wasthen applied to a Resource PHE column (1 ml, GE Healthcare Bio-Sciences)equilibrated beforehand with Buffer E. The column was washed with BufferE, and then a fraction containing the enzyme having the Ilehydroxylation activity was eluted with Buffer F (50 mM MES (pH 6.5), 10%of glycerol, 2 mM DTT, 1 mM EDTA, Complete (Roche)) by using a reverselinear concentration gradient of ammonium sulfate.

The outline of the isolation and purification of L-Ile hydroxylase aresummarized in Table 7.

TABLE 7 Summary of isolation and purification Specific Total Totalprotein activity activity Yield Fraction (mg) (U/mg) (U) (%) Cell-freeextract 678.173 3.8 2593.7 100.0 DEAE 73.324 17.8 1306.5 50.4 monoS3.639 96.5 351.0 13.5 Ammonium sulfate 0.578 209.5 121.1 4.7precipitation GPC 0.093 1222.8 113.4 4.4 Resource PHE 0.004 3694.9 13.20.5

Example 3 Characterization of L-Ile Hydroxalase

<1> Analysis by Electrophoresis

The purified sample obtained in Example 2 was analyzed by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (polyacrylamide gel:PAG Mini “Daiichi” 15/25 (13 wells) produced by Daiich Pure ChemicalsCo., Ltd., molecular weight standards: Prestained SDS-PAGE Standards,Low Range, produced by Bio-Rad). As a result, the enzyme is made up ofsubstantially uniform subunits each having a molecular weight of about31,000±20,000.

<2> Effects of Addition of Cofactors

The effects of cofactors on the production of 4-hydroxyisoleucine byhydroxylation of Ile were examined by using the purified enzyme. TheL-Ile hydroxylase prepared as described in Example 2 was used in a 100mM HEPES (pH 6) reaction mixture containing final concentrations of 5 mML-Ile and 5 mM of a cofactor, and the amount of 4-hydroxyisoleucinewhich was produced was measured. As shown in Table 8, Fe²⁺ and2-oxoglutaric acid were essential for the production of4-hydroxyisoleucine, and the amount of 4-hydroxyisoleucine produced wasmaximized by further adding ascorbic acid. Therefore, these datastrongly suggest that a dioxygenase might be involved in the productionof 4-hydroxyisoleucine by hydroxylation of L-isoleucine. This result wasthe same as the when using a cell lysate.

TABLE 8 Effect of various cofactors on the production of purified4-hydroxyisoleucine Extract Substrate (L-Ile) Cofactor HIL (mM) + − —0.00 + + — 0.00 + + α-KG 0.00 + + Ascorbate 0.00 + + Fe²⁺ 0.00 + +α-KG + ascorbate 0.00 + + α-KG + Fe²⁺ 0.02 + + Ascorbate + Fe²⁺ 0.00 + +α-KG + ascorbate + Fe²⁺ 0.26

<3> Optimum pH

By using L-Ile hydroxylase prepared as described in Example 2, pHdependency on the producing of the 4-hydroxyisoleucine was evaluated.The enzymatic reaction solution contained 5 mM L-Ile, 5 mM Fe²⁺, 5 mM2-oxoglutaric acid, 5 mM ascorbic acid and 200 mM GTA (pH 3 to 12). Thereaction temperature was 30° C. The relative amounts of4-hydroxyisoleucine produced at various pH values are shown in FIG. 8.These values are relative to the pH which resulted in the production ofthe maximum amount of 4-hydroxyisoleucine, which was set as 100%.Production of a large amount was confirmed at pH 4 to 8, and productionof the most 4-hydroxyisoleucine was confirmed at pH 5 to 8.

<4> Optimum Temperature

By using L-Ile hydroxylase prepared as described in Example 2, theoptimum temperature of the 4-hydroxyisoleucine production activity wasevaluated. The enzymatic reaction solution contained 5 mM Ile, 5 mMFe²⁺, 5 mM 2-oxoglutaric acid, 5 mM ascorbic acid, and 100 mM HEPES (pH6). The relative amounts of 4-hydroxyisoleucine at various temperaturesare shown in FIG. 9. These values are relative to the temperature whichresulted in the production of the maximum amount of 4-hydroxyisoleucine,which was set as 100%. High production was confirmed for the temperaturerange of 0 to 40° C.

<5> Temperature Stability

By using L-Ile hydroxylase prepared as described in Example 2, thetemperature stability during production of 4-hydroxyisoleucine wasevaluated. The enzyme solution at pH 7.0 was incubated at 0 to 70° C.for 1 hour, and then the Ile hydroxylation activity was measured. Theenzymatic reaction solution contained 5 mM Ile, 5 mM Fe²⁺, 5 mM2-oxoglutaric acid, 5 mM ascorbic acid, and 100 mM HEPES (pH 6), and thereaction temperature was 30° C. The relative temperature stability atvarious temperatures is shown in FIG. 10. These values are relative tothe storage temperature which produced the maximum amount ofhydroxyisoleucine, which was set as 100%. The enzyme was inactivated at60° C. or higher.

<6> Substrate Reaction Characteristics

By using L-Ile hydroxylase prepared as described in Example 2, thereaction characteristics for various amino acids were evaluated. Theenzyme solution and the reaction mixture were mixed, and then thereaction proceeded at 30° C. for 1 hour, and the production of newsubstances was evaluated by HPLC analysis. The enzymatic reactionsolution contained 5 mM amino acid, 5 mM Fe²⁺, 5 mM 2-oxoglutaric acid,5 mM ascorbic acid, and 100 mM HEPES (pH 6). In addition toL-isoleucine, the amino acids D-isoleucine, L-leucine, L-valine,L-glutamic acid, and L-lysine were also individually evaluated. Theproduced 4-hydroxyisoleucine was analyzed as in Example 1. The resultsare shown in Table 9. Like when using a cell lysate, production of a newsubstance could not be confirmed for amino acids other thanL-isoleucine. Therefore, these data suggest that the enzyme is anL-isoleucine-specific dioxygenase, and contributes to the production ofnaturally occurring HIL, as similarly shown by the results of theexamination using a cell lysate.

TABLE 9 Reactivity for each amino acid Product L-Isoleucine Produced(HIL) D-Isoleucine None L-Leucine None L-Valine None L-Glutamic acidNone L-Lysine None

<7> Effects of Inhibitors

By using L-Ile hydroxylase prepared as described in Example 2, theeffect of inhibitors on the production of 4-hydroxyisoleucine wereexamined. The enzymatic reaction solution contained 5 mM Ile, 5 mM Fe²⁺,5 mM 2-oxoglutaric acid, 5 mM ascorbic acid, and 100 mM HEPES (pH 6),the reaction temperature was 30° C., and the reaction time was 1 hour.The amount of 4-hydroxyisoleucine produced when 10 mM of each inhibitor(EDTA, Cu²⁺, Zn²⁺) was added to the reaction system was measured. Theproduction of 4-hydroxyisoleucine in the presence of the inhibitors isshown in Table 10. These values are relative to the 4-hydroxyisoleucineproduction observed with no inhibitor, which was set as 100%. Theisoleucine hydroxylation activity was lost in the presence of theinhibitors.

TABLE 10 Effect of inhibitors Inhibitor Relative activity (%) None 100EDTA 0 Cu²⁺ 0 Zn²⁺ 0

<8> N-terminus Amino Acid Sequence

L-Ile hydroxylase prepared as described in Example 2 was subjected toelectrophoresis as described in Example 3, transferred to a PVDFmembrane (sequi-Blot™ PVDF membrane, Bio-Rad), and used in PPSQ-10 (aprotein sequencer produced by Shimadzu Corporation). The followingN-terminal sequence of the enzyme was obtained by this method.

(SEQ ID NO: 5)  1 LysMetSerGlyPheSerIleGluGluLys 1011 ValHisGluPheGluSerLysGlyPheLeu 20

Example 4 Purification of IDO from Bacillus thuringiensis (2-e-2) Strain

Environmental microorganisms were screened and a unique microbepossessing α-ketoglutarate-dependent L-isoleucine dioxygenase activitywas found. It was identified as Bacillus thuringiensis and stocked asBacillus thuringiensis strain 2-e-2 (FERM BP-10688).

1.1. Cultivation conditions: The following cultivation medium was usedin the experiments:

CM1 (Tryptone 10 g/l; Yeast extract 10 g/l; pH 7.0 adjusted by NaOH);

CM6 (Tryptone 10 g/l; Yeast extract 40 g/l; pH 7.0 adjusted by NaOH).

1.2. Inoculum preparation: Bacillus thuringiensis strain 2-e-2 wascultivated overnight at 30° C. in CM1 medium supplemented with 90 mM KPibuffer (90 mM KH₂PO₄ (pH 7) adjusted with KOH). Then, glycerol was addedup to 20%. The resulting cell suspension was aliquoted in 1.9 ml vialsand stored at −70° C. until used.

1.3. Fermentation conditions: Bacillus thuringiensis strain 2-e-2 wasfermented in Marubischi fermenters. The following cultivation parameterswere used: starting culture volume: 600 ml; agitation: 800 rev/min; air:1:1; pH 7.0 was stabilized using 1H NaOH/HCl feeding. One vial of afrozen suspension from (1.2) above (1.9 ml) was used to inoculate 600 mlof CM6 medium. Strain 2-e-2 was cultivated for about 7.5 hours. Then,the cells were harvested by centrifugation and stored at −70° C. untilused.

1.4. IDO activity assay: Cells from 25-100 ml of the Bacillusthuringiensis 2-e-2 culture were harvested by centrifugation at 4° C.and re-suspended in 2 ml of buffer A (50 mM MOPS, 10% glycerol, 1 mMEDTA, 1 mM DTT, protease inhibitor, pH 7.2). The cells were disruptedusing a French-pressure cell (3× at 1000 Psi). The reaction mixture (50μl) contained 50 mM HEPES (pH 7.0), 5 mM Ile, 5 mM ascorbate, 10 mMFeSO₄, 5 mM α-ketoglutarate, and an aliquot of the protein preparation.The reaction was incubated at 34° C. for 40 min with shaking. Thesynthesized 4HIL was detected using TLC analysis. Thin-layer silica gelplate (10×15 cm) spotted with an aliquot (1-2 μ1) of the reactionsolution was developed with a developing solvent (2-propanol: acetone:ammonia: water=100:100:25:16), and 4HIL was detected with the ninhydrinreagent. HPLC analysis was carried out as described in Example 4.

1.5. Purification and Identification of IDO from Bacillus thuringiensis2-e-2.

All chromatographic procedures were carried out using ÄKTAbasic100system (Amersham Pharmacia Biotech). The purification protocol includesthe following steps. Frozen cells (62 g of weight biomass) were thawedand re-suspended in 150 ml buffer A [50 mM TRIZMA, 5% glycerol, 1 mMEDTA, 1 mM DTT, protease inhibitor, pH 7 adjusted with HCl].

Step 1: Cells were disrupted by 2 passages through the French pressurecell (max P=1000 Psi) followed by centrifugation (14000 g, 4° C., 20min) to remove cellular debris. The protein preparation was added to 150ml of the DEAE resin equilibrated with buffer A. The final suspensionwas incubated at 4° C. for about 10 minutes with low shaking. Then, theresin with the adsorbed protein was transferred to the column (26×30 cm)and a two-step protein elution was carried out. At first, the column waswashed with 50 mM NaCl in buffer A. Then, the IDO activity was elutedwith 250 mM NaCl in buffer A.

Step 2: The IDO activity was concentrated by ammonium sulphateprecipitation (at 1.9 M) and resuspended in about 3 ml of buffer B[50 mMTRIZMA, 5% glycerol, 1 mM EDTA, 1 mM DTT, 100 mM NaCl, pH 7].

Step 3: 1.5 ml of the preparation from Step 2 was applied to theSuperdex 200 HR 10/30A column equilibrated with buffer B. Isocraticelution was performed at a 0.5 ml/min flow rate. Active fractions werepooled.

Step 4: The protein preparation from step 3 was applied to a 1.6 mlSourse15Q column equilibrated with buffer A. The elution was carried outat a flow rate of 0.5 ml/min by liner gradient 0-0.5 M NaCl in buffer B(10 column volumes). Each 1 ml fraction was collected. Active fractionswere pooled.

As a result, IDO was purified about 120-fold (see Table 11). SDS-PAGE ofthe final protein preparation revealed only one major band (about 70% oftotal protein, see FIG. 12).

TABLE 11 Purification of IDO from Bacillus thuringiensis 2-e-2. SpecificTotal activity protein Total activity (nmoles/ Yield Purification Step(mg) (nmoles/min) min/mg) (%) fold Cell extract 3072 19046 6 100 1 DEAE(batch) 1500 18750 12.5 98 2 (NH₄)₂SO₄ 109 12514 115 65 19 Superdex 2006 1356 226 7 38 HR Source 15Q 0.03 21 700 0.1 117

1.6 Identification of IDO.

The major protein was extracted from the SDS-PAGE gel and analyzed bymass spectrometry analysis. The treatment of gels, trypsinolysis,protein extraction, and mass analysis by time-of-flight matrix-assistedlaser desorption-ionization (MALDI-TOF) were carried out according toprotocols described by Govorun, V. M. et al, (The proteome comparativeanalysis of Helicobacter pylori clinical isolates. Biochemistry (Mosc),68, 1, 42-49 (2003)). The protein was identified by the set of itsphotolytic peptide masses using Peptide Fingerprint option of Mascotsoftware (Matrix Science, USA). The analyzed protein was identified byMS-analysis as a putative RBTH_(—)06809 protein from the Bacillusthuringiensis (serovar israelensis; ATCC 35646) strain (FIG. 13).

Example 5 Cloning IDO Genes from Bacillus thuringiensis Strain 2-e-2 andBacillus thuringiensis (Serovar israelensis, ATCC 35646) Strains

The purified sample was electrophoresed in a SDS (15-25%) polyacrylamidegel. Protein transfer was performed for 1 h at 1 mA cm² at roomtemperature onto a polyvinylidenedifluoride membrane. After beingstained with CBB, the IDO band was cut out and subjected to automatedEdman degradation on the protein sequencer model PPSQ-10 (Shimadzu Co.Ltd., Kyoto, Japan). The first 20 N-terminal amino acids of the purifiedprotein were determined.

The N-terminal amino acid sequence of the 31-kDa polypeptide wasdetermined to be KMSGFSIEEKVHEFESKGFL (SEQ ID NO: 5). After a BLASTsearch, this amino acids sequence showed high homology with hypotheticalproteins of RBTH_(—)06809 from Bacillus thuringiensis (serovarisraelensis) ATCC 35646 and BC1061 from Bacillus cereus ATCC 14579 asshown in Table 1. Sequence analysis of the RBTH_(—)06809 ORF anddetermination of the N-terminal amino acid of the purified proteinindicate that there is only one potential IDO translation start becausethe classic SD sequence is located 8 by upstream of the ATG start codon(FIG. 14). However, Lys(7) is an N-terminal amino acid of the purifiedprotein. Therefore, N-terminal processing, or the cleavage of theN-terminal amino acids, is likely necessary for IDO activity. Seemingly,such cleavage is likely accomplished by a specific protease whichco-expresses with the IDO in Bacillus sp., but is not present in E.coli. Therefore, to produce mature IDO in E. coli, special recombinantplasmids were constructed.

2.1. Bacteria: Bacillus thuringiensis (serovar israelensis, ATCC 35646)strain was obtained from the Russian Collection of IndustrialMicroorganisms (VKPM), accession number B-197.

2.2 Construction of the pMW119-IDO(Lys, 32) plasmid: To constructpMW119-IDO(Lys, 32) the following procedures were carried out.

A 0.8 kb DNA fragment of the chromosome of the Bacillus thuringiensis(serovar israelensis, ATCC 35646) strain was amplified usingoligonucleotides SVS170 (SEQ ID No:3) and SVS169 (SEQ ID No:4) asprimers (for detail see FIG. 15), and the purified chromosomal DNA asthe template. The following PCR protocol was used: initial cycle for 30seconds at 94° C.; 4 cycles for 40 seconds at 94° C.; 30 seconds at 49°C.; 40 seconds at 72° C.; 35 cycles for 30 seconds at 94° C.; 30 secondsat 54° C.; 30 seconds at 72° C. The PCR-fragment was digested with BamHIand Sad endonucleases and then ligated into a pMW119 vector which hadbeen previously treated with the same restrictases.

2.3 Construction of the pMW119-IDO(Lys, 23) plasmid: A 0.8 kb DNAfragment of the chromosome of the Bacillus thuringiensis strain 2-e-2was amplified using oligonucleotides SVS170 (SEQ ID No:3) and SVS169(SEQ ID No:4) as primers (for detail see FIG. 15), and the purifiedchromosomal DNA as the template. The following PCR protocol was used:initial cycle for 30 seconds at 94° C.; 4 cycles for 40 seconds at 94°C.; 30 seconds at 49° C.; 40 seconds at 72° C.; 35 cycles for 30 secondsat 94° C.; 30 seconds at 54° C.; 30 seconds at 72° C. The PCR-fragmentwas digested with BamHI and Sad endonucleases and then ligated into apMW119 vector which had been previously treated with the samerestrictases.

Cells of E. coli strain TG1 were transformed with the ligation mixtures.The resulting clones were selected on a X-gal/IPTG agar-plate(blue/white test). Then, the IDO activity was tested in the crude celllysates of selected clones.

As a result, two clones, TG1 [pMW119-(Lys, 32)] and TG1 [pMW119-(Lys,23)], were selected. Corresponding plasmids were isolated and sequenceanalysis of the cloned BamHI-SacI fragments for each plasmid was carriedout (see FIG. 16, FIG. 17).

Analyses of the determined DNA sequences revealed discrepancy betweenthe cloned genes and the known RBTH_(—)06809 ORF (FIG. 18, FIG. 19). Inaddition, a point mutation in the regulatory region of IDO(Lys, 23) onthe plasmid pMW119 was found (FIG. 15 C) resulting in elimination of theTAA stop codon of the leader peptide (1) and prolongation of translationup to the TGA stop codon (leader peptide (2)) which overlaps with theATG start codon (see FIG. 15 A, C). An additional mutation was detectedin the “−2” position, where a C was substituted by an A (see FIG. 15,C).

2.4. The IDO activity assay in crude cell lysate of TG1[pMW119-IDO(Lys,23) and TG1[pMW119-IDO(Lys, 32): To investigate the IDO activity incrude cell lysates of recombinant E. coli strains, the followingprocedures were carried out. Cells from 5 ml of culture were harvestedby centrifugation at 4° C., re-suspended in 0.5 ml of buffer A*(50 mMTRIZMA, 5% glycerol, 1 mM EDTA, 1 mM DTT, pH 7 adjusted by HCl) anddisrupted by sonication at 4° C. The reaction mixture (50 μl) contained50 mM HEPES pH 7.0, 5 mM Ile, 0.5 mM α-ketoglutarate, 5 mM ascorbate, 5mM FeSO₄, and an aliquot of the protein preparation. The reaction wasincubated at 34° C. for 1 hour with shaking. The synthesized 4HIL wasdetected using TLC or HPLC analysis as described in Example 4. Theresults are summarized in Table 12.

TABLE 12 IDO activity Strain (nmoles/mg*min) TG1[pMW119] ND^(a))TG1[pMW119-IDO(Lys, 23)] 15 TG1[pMW119-IDO(Lys, 32)]  3 ^(a))ND—nondetected ((≦0.03 nmoles/mg*min)

Example 6 Biotransformation of L-isoleucine into 4HIL Using IDO

Cells of recombinant E. coli strains TG1[pMW119-IDO(Lys, 23)] andTG1[pMW119-IDO(Lys, 32)] were cultivated in LB medium supplemented withampicillin (100 mg/l), until an optical density of A₅₄₀=4-5 was reached(approximately 6 hours). After that, cells from 2 ml culture broth wereharvested by centrifugation and resuspended in 1 ml of MI50 solution(100 mM KH₂PO₄ (pH 7 adjusted by NaOH), NH₄Cl 20 mM, MgSO₄ 2 mM, CaCl₂0.1 mM, ampicillin 150 μg/ml, Ile 50 mM, 0.5 mM α-ketoglutarate,glycerol 1%, yeast extract—0.005 g/l).

Then, the cells were cultivated for about 12 hours. After that, theconcentration of 4HIL was analyzed by TLC (HPLC). The data aresummarized in Table 13. As shown in Table 12, cultivation ofTG1[pMW119-IDO(Lys, 23)] resulted in the production of a more 4HIL ascompared with TG1[pMW119-IDO(Lys, 32)].

TABLE 13 Ile 4HIL OD540 supplied obtained Yield^(b)) Strain 0 h 12 h(mM) (mM) (%) TG1[pMW119] 10 20 50 ND^(a)) — TG1[pMW119-IDO(Lys, 23)] 1020 50 7 14 TG1[pMW119-IDO(Lys, 32)] 10 20 50 5 10 ^(a))ND—non detected(≦0.02 mM) ^(b))Yield was calculated as (4HIL obtained/Ile supplied)*100

Example 7 HPLC Measurement of 4-hydroxy-L-isoleucine

HPLC analysis: High pressure chromatograph (Waters, USA) withspectrofluorometer 1100 series (Agilent, USA) was used. The chosendetection wave range: excitation wavelength at 250 nm, range of emissionwavelengths were 320-560 nm. The separation by the accq-tag method wasperformed in a Nova-Pak™ C18 150×3.9 mm, 4 μm column (Waters, USA) at+400° C. The injection volume of the sample was 5 μl. The formation ofamino acid derivatives and their separation was performed according toWaters manufacturer's recommendation (Liu, H. et al, J. Chromatogr. A,828, 383-395 (1998); Waters accq-tag chemistry package. Instructionmanual. Millipore Corporation, pp. 1-9 (1993)). To obtain amino acidderivatives with 6-aminoquinolil-N-hydroxysuccinymidyl carbamate, theAccq-Fluor™ kit (Waters, USA) was used. The analysis by the accq-tagmethod was performed using concentrated Accq-tag Eluent A (Waters, USA).All solutions were prepared using Milli-Q water, and standard solutionswere stored at +4° C.

Example 8 Cloning of ido Genes from Bacillus cereus ATCC 14597, B.thuringiensis AKU238, and B. weihenstephanensis KBAB4

(1) Preparation of Chromosomal DNA

Bacillus cereus ATCC 14579, B. thurigiensis AKU238, and B.weihenstephanensis KBAB4 were each cultivated overnight at 28° C. in 5ml of LB medium (pre-culture). By using 1.5 ml of the culture broth as aseed, a main culture was carried out in 50 ml of LB medium. Aftercultivation up to the logarithmic growth phase, cells were harvestedfrom 50 ml of the culture broth by centrifugation (12000×g, 4° C., 15min). From these cells, chromosomal DNA was prepared according to aknown method.

(2) Isolation of the ido Genes by PCR

Based on the published genomic sequence information about B. cereus ATCC14579 (GenBank accession No. AE016877), the following primers weresynthesized:

(SEQ ID NO: 10) CATATGGAGGTTTTTATAATGACGTTTGTT (SEQ ID NO: 11)CTCGAGTTTTGTCTCCTTATAAGAAAATGT

By using the prepared primers and chromosomal DNA of B. cereus ATCC14578 as the template, amplification by PCR was carried out withPrimeSTAR (TaKaRa) under the following conditions: 30 cycles for 10seconds at 98° C., 15 seconds at 52° C. and 1 minute at 72° C.

The PCR product was subjected to agarose gel electrophoresis and afragment of about 750 bp was amplified. The DNA fragment was collected,digested with NdeI and XhoI endonucleases, and cloned into theexpression vector pET21b (Novagene) which had been digested with thesame endonucleases. Then, the nucleotide sequence was determined and thecorresponding amino acid sequence was deduced (SEQ ID NOS: 12 and 13).As a result, it was confirmed that the ido gene is homologous to theobtained DNA fragment. The homology at nucleotide sequence level was 98%relative to both the ido gene from B. thuringiensis israelensis ATCC35646 and the ido gene from B. thuringiensis 2-e-2.

The ido gene from B. thuringiensis AKU238 was cloned using the sameprimers (SEQ ID NOS: 14 and 15) which were used to clone the ido genefrom B. thuringiensis 2-e-2 (SEQ ID NOS: 16 and 17). The homology atnucleotide sequence level was 98% relative to both the ido gene from B.thuringiensis israelensis ATCC 35646 and the ido gene from B.thuringiensis 2-e-2.

(SEQ ID NO: 14) CATATGAAAATGAGTGGCTTTAGCATAGAA (SEQ ID NO: 15)CTCGAGTTTTGTCTCCTTATAAGAAAATGT

The ido gene from B. weihenstephanensis KBAB4 was cloned using primersbased on the published genomic sequence (GenBank accession No.NZ_AAOY01000001 (SEQ ID NOS:18 and 19). PCR was used to clone an idogene, which was sequenced in a similar manner to the above (SEQ ID NOS:20 and 21). The homology at nucleotide sequence level was 78% relativeto both the ido gene from B. thuringiensis israelensis ATCC 35646 andthe ido gene from B. thuringiensis 2-e-2.

(SEQ ID NO: 18) CATATGCTAACAACAGTTTCTAATAAGACA (SEQ ID NO: 19)CTCGAGTTTTGGCTCCTTATAAGAAAACGT

Example 9 Expression of ido Genes in E. coli and Production of HIL fromIle

(1) Expression of ido Genes in E. coli

The plasmid expressing the ido gene from B. cereus ATCC 14579, B.thuringiensis AKU238, or B. weihenstephanensis KBAB4, which wasconstructed in Example 8, was introduced into E. coli Rosetta2 (DE3),and the transformants were cultivated with shaking in LB mediumsupplemented with 50 μg/ml ampicilin (preculture). By seeding thepreculture broth in 50 ml LB medium at 1%, the main culture was carriedout at 37° C. 2 hours after the start of the cultivation, IPTG was addedto a final concentration of 1 mM, and the cultivation was carried outfor an additional 3 hours. After completion of the cultivation, thecells were harvested, washed, suspended in 1 ml of 20 mM Tris-HCl (pH7.6), and disrupted with a sonicator (INSONATOR 201M, KUBOTA). Thelysate was centrifuged at 15000 rpm for 10 minutes, and the supernatantwas used as a crude enzyme solution.

(2) Production of HIL from Ile and the Crude Enzyme Solution

By using the crude enzyme solution prepared in (1), the conversion ofIle to HIL was measured. The reaction mixture is described below. Thereaction mixture was reacted with shaking (300 rpm) at 28° C. for 3hours, and then the amount of HIL produced was determined by HPLC. Theresults are shown in Table 14.

Reaction mixture: Cell lysate (Ultrasonic disruption) 500 μl 2% Ile 100μl 1M α-KG 5 μl 1M Ascorbate 5 μl 0.1M FeCl₂ 10 μl 1M KPB (pH 6) 100 μlTotal 1 ml

TABLE 14 Amount of HIL produced from Ile HIL (μM) B. thuringiensisisraelensis ATCC35646 2150 B. thuringiensis 2e2 2321 B. thuringiensisAKU238 2016 B. cereus ATCC 14579 90 B. weihenstephanensis KBAB4 3116control (pET21b) 11

When cells expressing the ido gene from B. cereus ATCC 14579, and whenthe plasmid expressing the ido gene from B. thuringiensis AKU238 or B.weihenstephanensis KBAB4, were used, HIL was clearly produced. Thus, itwas confirmed that these genes are useful in HIL production.

Explanation of Sequences

1: Nucleotide sequence of IDO gene from B. thuringiensis strain 2-e-2

2: Amino acid sequence of IDO from B. thuringiensis strain 2-e-2

3: Primer sys 170; for amplification of IDO gene

4: Primer sys 169; for amplification of IDO gene

5: N-Terminal sequence of IDO from B. thuringiensis strain 2-e-2

6: IDO conserved sequence among Bacillus genus

7: Nucleotide sequence of IDO gene from B. thuringiensis strain ATCC35646

8: Amino acid sequence of IDO from B. thuringiensis strain ATCC 35646

9: 16S rDNA nucleotide sequence of B. thuringiensis strain 2-e-2

10: Primer for amplification of IDO gene from B. cereus ATCC 14579

11: Primer for amplification of IDO gene from B. cereus ATCC 14579

12: Nucleotide sequence of IDO gene from B. cereus ATCC 14579

13: Amino acid sequence of IDO from B. cereus ATCC 14579

14: Primer for amplification of IDO gene from B. thuringiensis AKU238

15: Primer for amplification of IDO gene from B. thuringiensis AKU238

16: Nucleotide sequence of IDO gene from B. thuringiensis AKU238

17: Amino acid sequence of IDO from B. thuringiensis AKU238

18: Primer for amplification of IDO gene from B. weihenstephanensisKBAB4

19: Primer for amplification of IDO gene from B. weihenstephanensisKBAB4

20: Nucleotide sequence of IDO gene from B. weihenstephanensis KBAB4

21: Amino acid sequence of IDO from B. weihenstephanensis KBAB4

INDUSTRIAL APPLICABILITY

According to the present invention, a method is provided for producing4-hydroxyisoleucine using an enzyme derived from a microorganism andcatalyzing production of 4-hydroxyisoleucine by direct hydroxylation ofisoleucine. The present invention is extremely useful in the fields ofpharmaceuticals and food.

The L-isoleucine dioxygenase described herein is a novel dioxygenasethat catalyzes the hydroxylation of L-isoleucine, and may be used tosynthesize (2S,3R,4S)-4-hydroxy-L-isoleucine. This compound is useful asa component of pharmaceutical compositions with insulinotropic activity.

1. An isolated DNA selected from the group consisting of: (a) a DNAcomprising the nucleotide sequence of SEQ ID No: 1; (b) a DNA thathybridizes under stringent conditions with a DNA comprising a nucleotidesequence complementary to the nucleotide sequence of SEQ ID No: 1, andwherein said stringent conditions comprise washing at a saltconcentration of 0.1×SSC and 0.1% SDS at 37° C., and wherein said DNAencodes a protein having L-isoleucine dioxygenase activity; (c) a DNAthat encodes a protein comprising the amino acid sequence of SEQ ID No:2; (d) a DNA that encodes a protein comprising the amino acid sequenceof SEQ ID No: 2, but which includes one to 13 substitutions, deletions,insertions, additions, or inversions of one to 13 amino acids andwherein said protein has L-isoleucine dioxygenase activity; and (e) aDNA that encodes a protein comprising an amino acid sequence that is atleast 98% homologous to the amino acid sequence of SEQ ID NO: 2, and hasL-isoleucine dioxygenase activity.
 2. A recombinant DNA obtained byligating the DNA according to claim 1 with a vector DNA.
 3. A celltransformed with the recombinant DNA according to claim
 2. 4. A processfor producing a protein having L-isoleucine dioxygenase activity, theprocess comprising: A) cultivating the cell according to claim 3 in amedium, and B) collecting the protein with L-isoleucine dioxygenaseactivity from the medium, cells, or both.